(19)
Europaisches Patentamt
European Patent Office
Office europeen des brevets
(12) EUROPEAN PATENT APPLICATION
published in accordance with Art. 158(3) EPC
(43) Date of publication:
(51) Intel. 7 : C12N 15/09, C12N 15/62,
lo.U/.zUUo Id U I lei ID zUUo/zs
C07K 1 6/28, A61 K 39/395
^1 j Application numuer. uia/ooo^.u
(86) International application number:
Date Or tiling. 22.10.2001
PCT/JP01/09260
(87) International publication number:
WO 02/033073 (25.04.2002 Gazette 2002/17)
(84) Designated Contracting States:
• TSUCHIYA, Masayuki,
AT BE CH CY DE DK ES Fl FR GB GR IE IT LI LU
c/o CHUGAI SEIYAKU K.K.
MC NL PT SE TR
Gotemba-shi, Shizuoka 412-8513 (JP)
Designated Extension States:
• UNO, bhinsuke,
AL LT LV MK RO SI
c/o CHUGAI SEIYAKU KABUSHIKI KAISHA
Gotemba-shi, Shizuoka 412-8513 (JP)
(30) Priority: 20.10.2000 JP 2000321821
• OHTOMO, Toshihiko,
20.10.2000 JP 2000321822
c/o CHUGAI SEIYAKU KABUSHIKI K.
12.03.2001 WOPCT/JP01/01912
Gotemba-shi, Shizuoka 412-8513 (JP)
17.04.2001 WOPCT/JP01/03288
• YABUTA, Naohiro,
12.09.2001 JP 2001277314
c/o CHUGAI SEIYAKU KABUSHIKI K.
Niihari-gun, Ibaraki 300-4101 (JP)
(71) Applicant: CHUGAI SEIYAKU KABUSHIKI
• TSUNODA, Hiroyuki,
KAISHA
c/o CHUGAI SEIYAKU KABUSHIKI K.
Tokyo, 115-8543 (JP)
Niihari-gun, Ibaraki 300-4101 (JP)
(72) Inventors:
(74) Representative: HOFFMANN EITLE
• FUKUSHIMA, Naoshi, c/o CHUGAI SEIYAKU K.K.
Patent- und Rechtsanwalte
Gotemba-shi, Shizuoka 412-8513 (JP)
Arabellastrasse 4
81925 Munchen (DE)
(54) DEGRADED AGONIST ANTIBODY
(57) The invention relates to a modified antibody
which contains two or more H chain V regions and two
or more L chain V regions of monoclonal antibody and
can transduce a signal into cells by crosslinking a cell
surface molecule(s) to thereby serve as an agonist. The
modified antibody can be used as a signal transduction
agonist and, therefore, useful as a preventive and/or
remedy for various diseases such as cancer, inflamma-
tion, hormone disorders and blood diseases.
CO
CD
CM
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Printed by Jouve, 75001 PARIS (FR)
EP 1 327 681 A1
Description
TECHNICAL FIELD
s [0001] This invention relates to modified antibodies containing two or more H chain V regions and two or more L
chain V regions of a monoclonal antibody which show an agonist activity by crosslinking a cell surface molecule(s) or
intracellular molecule(s). The modified antibodies have an agonist activity of transducing a signal into cells by crosslink-
ing a cell surface molecule(s) and are useful as a medicine for various purposes.
10 BACKGROUND ART
[0002] JP-A 9-295999 discloses the preparation of a specific monoclonal antibody using a splenic stromal cell line
as a sensitizing antigen aiming at developing specific antibodies that can recognize the aforementioned splenic stromal
cells and the preparation of novel monoclonal antibodies that recognize mouse Integrin Associated Protein (mouse
15 |AP) as an antigen. JP-A. 9-295999 also discloses that the monoclonal antibodies are capable of inducing apoptosis
of myeloid cells.
[0003] W099/1 297 discloses monoclonal antibodies whose antigen is human Integrin Associated Protein (hereinafter
referred to as human IAP; amino acid sequence and nucleotide sequence thereof are described in J. Cell Biol., 123,
485-496, 1 993; see also Journal of Cell Science, 1 08, 341 9-3425, 1 995) and which are capable of inducing apoptosis
20 of human nucleated blood cells (myeloid cell and lymphocyte) having said human IAP. These monoclonal antibodies
are referred to antibody MABL-1 and antibody MABL-2, and hybridomas producing these antibodies are also referred
to MABL-1 (FERM BP-6100) and MABL-2 (FERM BP-6101), respectively.
[0004] Japanese Patent Application 1 1 -63557 describes the preparation of single chain Fvs having single chain Fv
regions from the monoclonal antibodies whose antigen is human IAP. The single chain Fvs are capable of inducing
25 apoptosis of nucleated blood cells having human IAP.
[0005] The monoclonal antibody recognizing IAP as an antigen induces apoptosis of nucleated blood cells having
human IAP, but it also causes hemagglutination in vitro . It indicates that the administration of a large amount of the
monoclonal antibody recognizing IAP as an antigen may result in a side effect such as hemagglutination.
[0006] The inventors made intensive research for utilizing the monoclonal antibodies against human IAP as thera-
30 peutic agent of blood diseases and obtained single chain Fvs having the single chain Fv region capable of inducing
apoptosis of nucleated blood cells having human IAP
[0007] On the other hand modified antibodies, especially antibodies with lowered molecular size, for example, single
chain Fvs were developed to improve permeability into tissues and tumors by lowering molecular size and to produce
by a recombinant method. Recently the dimers of single chain Fvs, especially bispecific-dimers have been used for
35 crosslinking cells. Typical examples of such dimers are hetero-dimers of single chain Fvs recognizing antigens of
cancer cells and antigens of host cells like NK cells and neutrophils (Kipriyanov et al., Int. J. Cancer, 77, 9763-9772,
1998). They were produced by construction technique of single chain Fv as modified antibodies, which are more ef-
fective in treating cancers by inducing intercellular crosslinking. It has been thought that the intercellular crosslinking
is induced by antibodies and their fragments (e.g. Fab fragment), bispecific modified antibodies and even dimers of
40 single chain Fvs, which are monospecific.
[0008] As antibodies capable of transducing a signal by crosslinking a cell surface molecule(s), there are known an
antibody against EPO receptor involved in cell differentiation and proliferation (JP-A 2000-95800), an antibody against
MuSK receptor (Xie et al., Nature Biotech. 15, 768-771, 1997) and others. However there have been no reports on
modified antibodies with lowered molecular size.
45 [0009] Noticing that single chain Fv monomers derived from antibody MABL-1 and antibody MARL-2 do not induce
apoptosis of cells while single chain Fv dimers induce apoptosis of cells having IAP, the inventors discovered that they
crosslink (dimerize) IAP receptor on cell surface, thereby a signal is transduced into the cells and, as a result, apoptosis
is induced. This suggests that monospecific single chain Fv dimers crosslink a cell surface molecule(s) (e.g. receptor)
and transduce a signal like a ligand, thereby serving as an agonist.
so Focusing on the intercellular crosslinking, it was discovered that the above-mentioned single chain Fv dimers do not
cause hemagglutination while the above-mentioned monoclonal antibodies do. The same result was also observed
with single chain bivalent antibodies (single chain polypeptides containing two H chain V regions and two L chain V
regions). This suggests that monoclonal antibodies may form intercellular crosslinking while modified antibodies like
single chain Fv dimers and single chain bivalent antibodies crosslink a cell surface molecule(s) but do not form inter-
55 cellular crosslinking.
[0010] Based on those observations the inventors have newly discovered that modified antibodies such as single
chain Fv dimers and single chain bivalent antibodies crosslink a cell surface molecule(s) or intercellular molecule(s)
of the same cell, in addition to known intercellular crosslinking, and are suitable as a ligand to the molecule(s) (especially
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EP 1 327 681 A1
as a ligand which mimics the action of natural ligand).
[001 1] Discovering further that an antibody molecule (whole IgG) can be modified into single chain Fv dimers, single
chain bivalent antibodies and the like which crosslink a cell surface molecule(s), thereby reducing side effects caused
by intercellular crosslinking and providing new medicines inducing only desired effect on the cell, the inventors com-
s pleted the invention. The modified antibodies of the invention have remarkably high activity compared with natural
ligands such as TPO, EPO or G-CSF, or whole antibodies (IgG) having the same V region as the modified antibodies.
They have an improved permeability into tissues due to the lowered molecular size compared with antibody molecules
and the lack of constant regions.
10 DISCLOSURE OF INVENTION
[0012] An object of this invention is to provide low molecular-sized agonist modified antibodies which contain two or
more H chain V regions and two or more L chain V regions of monoclonal antibodies and have an agonist action by
crosslinking a cell surface molecule(s) or intracellular molecule(s).
15 [0013] Therefore, this invention relates the modified antibodies which contain two or more H chain V regions and
two or more L chain V regions, preferably 2 to 6 each, especially preferably 2 to 4 each, most preferably two each,
and show an agonist activity by crosslinking a cell surface molecule(s) or intracellular molecule(s).
[0014] The "modified antibodies" in the specification mean any substances which contain two or more H chain V
regions and two or more L chain V regions, wherein said V regions are combined directly or via linker through covalent
20 bond or non-covalent bond. For example, polypeptides and compounds produced by combining each V region of
antibody through a peptide linker or a chemical crosslinking agent and the like. Two or more H chain V regions and
two or more L chain V regions used in the invention can be derived from the same antibody or from different antibodies.
[0015] Preferable examples of modified antibodies of the invention are multimerssuch as dimers, trimers ortetramers
of single chain Fv containing an H chain V region and an L chain V region, or single chain polypeptides containing two
25 or more H chain V regions and two or more L chain V regions. When the modified antibodies of the invention are
multimers of single chain Fv such as dimers, trimers, tetramers and the like containing an H chain V region and an L
chain V region, it is preferable that the H chain V region and L chain V region existing in the same chain are not
associated to form an antigen-binding site.
[0016] More preferable examples are dimers of the single chain Fv which contains an H chain V region and an L
30 chain V region, or a single chain polypeptide containing two H chain V regions and two L chain V regions. The H chain
V region and L chain V region are connected preferably through a linker in the modified antibodies.
[0017] "Agonist action" in the specification means a biological action occurring in the cell(s) into which a signal is
transduced by crosslinking a cell surface molecule(s) or intracellular molecule(s), for example, apoptosis induction,
cell proliferation induction, cell differentiation induction, cell division induction or cell cycle regulation action.
35 [0018] ED50 of the agonist action in the invention is determined by known methods for measuring agonist action.
Examples are to detect agonist specific cell death or cell proliferation, to detect expression of proteins specific to cell
differentiation (e.g. specific antigens) or to measure a kinase activity specific to cell cycle. ED50 is a dose needed for
achieving 50% reaction of the maximum activity set as 100% in the dose-reaction curve.
[0019] Preferable modified antibodies of the invention have an agonist action (ED50) equivalent to or better than
40 that of an antibody having the same antigen-binding region as the modified antibody, namely the whole antibody like
IgG (hereinafter "parent antibody" ) having the same pair of H chain V region and L chain V region as the pair of H
chain V region and L chain V region forming antigen-biding region of the modified antibody. More preferable are those
having an agonist action (ED50) more than two times higher than that of parent antibody, further preferably more than
5 times, most preferably more than 10 times. The invention includes modified antibodies with an agonist action con-
45 taining H chain V region and L chain V region forming the same antigen-binding region as parent antibody which binds
to target cell surface molecule(s) or intracellular molecule(s) but has no agonist action to the molecule.
[0020] The compounds containing two or more H chain V regions and two or more L chain V regions of the invention
can be any compounds which contain two or more H chain V regions and two or more L chain V regions of antibody
and show an agonist action (ED50) equivalent to or better than that of a natural ligand binding to a cell surface molecule
so (s) or intracellular molecule(s). Preferable are those having an agonist action (ED50) more than two times higher than
that of a natural ligand, more preferably more than 5 times, most preferably more than 10 times.
[0021] The "compounds" mentioned here include not only modified antibodies of the invention but also any com-
pounds containing two or more, preferably from 2 to 6, more preferably from 2 to 4, most preferably 2 antigen-binding
regions such as whole antibodies or F(ab') 2 .
55 [0022] The modified antibodies or compounds of the invention containing two or more H chain V regions and two or
more L chain V regions of antibody have preferably no substantial intercellular adhesion action. When the H chain V
region and L chain V region of the modified antibodies of the invention are derived from the same antibody, those are
preferable with an intercellular adhesion action (ED50) not more than 1/10 compared with the original antibody.
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[0023] ED50 of intercellular adhesion action in the invention is determined by known methods for measuring agonist
action, for example, by the measurement of agglomeration action of cells expressing said cell surface molecule such
as hemagglutination test.
[0024] The invention relates to DNAs which code for the modified antibodies.
s [0025] The invention relates to animal cells or microorganisms which produce the modified antibodies.
[0026] The invention relates to use of the modified antibody as an agonist.
[0027] The invention relates to a method of transducing a signal into cells by crosslinking cell surface molecule or
intracellular molecule using the modified antibody and thereby inducing an agonist action of cells such as apoptosis
induction, cell proliferation induction, cell differentiation induction, cell division induction or cell cycle regulation action.
w [0028] The invention relates to a medicine containing the modified antibody.
[0029] The invention relates to use of the modified antibody as a medicine.
[0030] The invention relates to a method of screening or measuring the modified antibody, which contains two or
more H chain V regions and two or more L chain V regions of antibody and shows an agonist action by crosslinking
cell surface molecule or intracellular molecule, that comprises 1 ) to prepare a modified antibody containing two or more
15 H chain V regions and two or more L chain V regions of antibody and binding specifically to said molecule, 2) to contact
the modified antibody with cells expressing said molecule and 3) to measure an agonist action which occurs in the
cells caused by crosslinking said molecule. The method of measurement is useful for the quality control in producing
the modified antibodies of the invention as a medicine and other purposes.
[0031] The above-mentioned single chain Fv dimer includes a dimer by non-covalent bond, a dimer by a covalent
20 bond through a crosslinking radical and a dimer through a crosslinking reagent (an antibody, an antibody fragment, or
bivalent modified antibody). Conventional crosslinking radicals used for crosslinking peptides can be used as the
crosslinking radicals to form the dimers. Examples are disulfide crosslinking by cysteine residue, other crosslinking
radicals such as C 4 - C 10 alkylene (e.g. tetramethylene, pentamethylene, hexamethylene, heptamethylene and octam-
ethylene, etc.) or C 4 - C 10 alkenylene (cis/trans -3-butenylene, cis/trans-2-pentenylene, cis/trans-3-pentenylene, cis/
25 trans-3-hexenylene, etc.).
[0032] Moreover, the crosslinking reagent which can combine with a single chain Fv is, for example, an amino acid
sequence which can optionally be introduced into Fv, for example, an antibody against FLAG sequence and the like
or a fragment thereof, or a modified antibody originated from the antibody, for example, single chain Fv.
[0033] The invention also relates to a method of inducing an agonist action to cells by administering the first ligand
30 and the second ligand which combine with a cell surface molecule(s) or intracellular molecule(s), and administering a
substance which combine with the first and the second ligands and crosslink the first and second ligands. The first
ligand and the second ligand can be any things which contain a biding site to said molecule and can induce an agonist
action by being crosslinked. Preferable examples are monovalent modified antibodies, such as the same or different
single chain Fv monomer, a fragment of antibody etc. The substance to crosslink the above-mentioned ligand can be
35 any things that induce an agonist action to the cells by crosslinking the first ligand and the second ligand. Preferable
examples are antibodies, fragments of antibodies, (Fab) 2 or bivalent modified antibodies. Examples of bivalent anti-
bodies are (Fab) 2 , dimers of single chain Fv containing one H chain V region and one L chain V region and single
chain polypeptides containing two H chain V regions and two L chain V regions. The method is effective for exploring
receptors that transduce a signal into cells by crosslinking, is expected to be employed for DDS to deliver a medicine
40 to target cells and is also useful as a drug administration system which suppresses side effect and allows a medicine
to become effective at desired time and for desired period.
[0034] The modified antibodies of this invention can be any things which contain L chain V region and H chain V
region of antibody (e.g. antibody MABL- 1 , antibody MABL-2, antibody 1 2B5, antibody 12E10 etc.) and which specif-
ically recognize the cell surface molecule(s) or intracellular molecule(s), for example, a protein (a receptor or a protein
45 involved in signal transduction), or a sugar chain of the above-mentioned protein or of a cell membrane protein and
crosslink said cell surface molecule(s), thereby transduce a signal into cells. Modified antibodies in which a part of
amino acid sequence of V region has been altered are included.
[0035] Depending upon the characteristics of cell surface molecule or intracellular molecule to be combined, for
example, the structure of molecule or the action mechanism, the modified antibodies can be mono-specific or multi-
50 specific like bi-specific. When the modified antibody is combined with a receptor molecule which homodimerizes and
transduces a signal into the cells (e.g. erythropoietin receptor, thrombopoietin receptor, G-CSF receptor, SCF receptor,
EGF receptor, IAP(CD47) and the like), mono-specific modified antibody is preferable. When it is combined with a
receptor molecule which heterodimerizes and transduces a signal into the cells (e.g. IL-6 receptor, LIF receptor, IL-11
receptor), bi-specific modified antibody is preferable. When it is combined with a receptor molecule which heterotrimer-
55 izes and transduces a signal into the cells (e.g. IL-2 receptor, CNTF receptor, OSM receptor), tri-specific modified
antibody is preferable. A method for producing bi-specific single chain Fv dimers is described in WO9413804 and the
like.
[0036] The present invention also relates to modified antibodies whose H chain V region and/or L chain V region is
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EP 1 327 681 A1
H chain V region derived from human antibody and/or L chain V region derived from human antibody. The H chain V
region and/or L chain V region derived from human antibody can be obtained by screening human nomoclonal anti-
body's library as described in W099/1 0494. The H chain V region and L chain V region derived from human monoclonal
antibodies are also included.
s [0037] The present invention further relates to modified antibodies whose H chain V regions and/or L chain V regions
are humanized H chain V regions and/or humanized L chain V regions. Specifically, the humanized modified antibodies
consist of the humanized L chain V region which comprises framework regions (FR) derived from an L chain V region
of human monoclonal antibody and complementarity determining regions (hereinafter "CDR") derived from an L chain
V region of non-human mammalian (e.g. mouse, rat, bovine, sheep, ape) monoclonal antibody and/or the humanized
10 H chain V region which comprises FR derived from an H chain V region of human monoclonal antibody and CDR
derived from an H chain V region of non-human mammalian (e.g. mouse, rat, bovine, sheep, ape) monoclonal antibody.
In this case, the amino acid sequence of CDR and FR may be partially altered, e.g. deleted, replaced or added.
[0038] H chain V regions and/or L chain V regions of the modified antibodies of the invention can be H chain V
regions and/or L chain V regions derived from monoclonal antibodies of animals other than human (such as mouse,
15 rat, bovine, sheep, ape, chicken and the like). In this case, the amino acid sequence of CDR and FR may be partially
altered, e.g. deleted, replaced or added.
[0039] The invention also relates to DNAs encoding the various modified antibodies as mentioned above and genetic
engineering techniques for producing recombinant vectors comprising the DNAs.
[0040] The invention also relates to host cells transformed with the recombinant vectors. Examples of host cells are
20 animal cells such as human cells, mouse cells or the like and microorganisms such as E. coli , Bacillus subtilis , yeast
or the like.
[0041] The invention relates to a process for producing the modified antibodies, which comprises culturing the above-
mentioned hosts and extracting the modified antibodies from the culture thereof.
[0042] The present invention further relates to a process for producing adimer of the single chain Fv which comprises
25 culturing host animal cells producing the single chain Fv in a serum-free medium to secrete the single chain Fv into
the medium and isolating the dimer of the single chain Fv formed in the medium.
[0043] The present invention also relates to the use of the modified antibodies as an agonist. That is, it relates to
the signal-transduction agonist which comprises as an active ingredient the modified antibody obtained as mentioned
above. Since the modified antibodies used in the invention are those that crosslink a cell surface molecule(s) or intra-
30 cellular molecule(s) and induce signal transduction, the molecule can be any molecule that is oligomerized, e.g. dimer-
ized, by combining with the ligand and thereby transduce a signal into cells.
[0044] Such cell surface molecule includes hormone receptors and cytokine receptors. The hormone receptor in-
cludes, for example, estrogen receptor. The cytokine receptor and the like include hematopoietic factor receptor, lym-
phokine receptor, growth factor receptor, differentiation control factor receptor and the like. Examples of cytokine re-
35 ceptors are erythropoietin (EPO) receptor, thrombopoietin (TPO) receptor, granulocyte colony stimulating factor
(G-CSF) receptor, macrophage colony stimulating factor (M-CSF) receptor, granular macrophage colony stimulating
factor (GM-CSF) receptor, tumor necrosis factor (TNF) receptor, interleukin-1 (IL-1) receptor, interleukin-2 (IL-2) re-
ceptor, interleukin-3 (IL-3) receptor, interleukin-4 (IL-4) receptor, interleukin-5 (IL-5) receptor, interleukin-6 (IL-6) re-
ceptor, interleukin-7 (IL-7) receptor, interleukin-9 (IL-9) receptor, interleukin-1 0 (IL-1 0) receptor, interleukin-1 1 (IL-11)
40 receptor, interleukin-1 2 (IL-1 2) receptor, interleukin-1 3 (IL-13) receptor, interleukin-1 5 (IL-1 5) receptor, interferon-alpha
(IFN-alpha) receptor, interferon-beta (IFN-beta) receptor, interferon-gamma (IFN-gamma) receptor, growth hormone
(GH) receptor, insulin receptor, blood stem cell proliferation factor (SCF) receptor, vascular endothelial growth factor
(VEGF) receptor, epidermal cell growth factor (EGF) receptor, nerve growth factor (NGF) receptor, fibroblast growth
factor (FGF) receptor, platelet-derived growth factor (PDGF) receptor, transforming growth factor-beta (TGF-beta) re-
45 ceptor, leukocyte migration inhibitory factor (LIF) receptor, ciliary neurotrophic factor (CNTF) receptor, oncostatin M
(OSM) receptor, Notch family receptor and the like.
[0045] The intracellular surface molecule includes TAK1 , TAB1 and the like. TAK1 and TAB1 act in signal transduction
pathway of TGF-p, activate MAP kinase by forming hetero-dimer and transduce a series of signals. Many cancer cells
have mutation of TGF-fS receptor, which represses the growth of cancer, and, therefore, the signal of TGF-p is not
so transduced. The modified antibodies, which can transduce a signal by crosslinking TAK1 and TAB1, can induce the
signal of TGF-p through an agonistic action by combining with TAK1/TAB1 . Such modified antibodies of the invention
can inhibit the growth of TGF-P resistant cancer cells and provide a new method for cancer therapy. Other examples
of intracellular molecule are transcription factor E2F homo-dimer and E2F/DP1 hetero-dimer having cell proliferation
action. The modified antibodies of the invention can induce an agonist action also on those molecules, and therefore
55 can be used for the treatment of various cell-proliferation-related diseases. The modified antibodies of the invention
can induce an agonist action by crosslinking intracellular factor involved in apoptosis-induction-related signal trans-
duction and therefore can induce apoptosis cell death of cancer cells or autoimmune-disease-related cells.
[0046] To achieve the interaction of the modified antibodies of the invention with intracellular molecule, peptides with
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EP 1 327 681 A1
cell-membrane-permeation-ability (e.g. Pegelin, Penetratin) can be used to transport the modified antibodies into the
cells (Martine Mazel et al, Doxorubicin-peptide conjugates overcome multidrug resistance. Anticancer Drugs 2001,
12, Dccrossi D. et al., The third helix of the antennapedia homeodomain translocates through biological membranes,
J. Biol. Chem. 1994, 269, 10444-10450).
s [0047] Therefore, the pharmaceutical preparations containing the agonist modified antibody as an active ingredient
are useful as preventives and/or remedies etc. for various diseases such as cancers, inflammation, hormone disorders,
blood diseases and autoimmune diseases.
[0048] Oligomers which can be formed by receptor proteins can be homo-oligomers or hetero-oligomers, and any
oligomers such as dimers, trimers and tetramers. It is known for example that erythropoietin receptor, thrombopoietin
10 receptor, G-CSF receptor, SCF receptor, EGF receptor and the like form homo-dimers, that IL-6 receptor, LIF receptor
and IL-11 receptor form hetero-dimers and that IL-2 receptor, CNTF receptor, OSM receptor form hetero-trimers.
[0049] The modified antibodies of the present invention comprise two or more H chain V regions and two or more L
chain V regions derived from monoclonal antibodies. The structure of the modified antibodies may be a dimer of single
chain Fv comprising one H chain V region and one L chain V region or a polypeptide comprising two H chain V regions
15 and two L chain V regions. In the modified antibodies of the invention, the V regions of H chain and L chain are preferably
linked through a peptide linker which consists of one or more amino acids. The resulting modified antibodies contain
variable regions of antibodies and bind to the antigen with the same specificity as that of the original monoclonal
antibodies.
20 H chain V region
[0050] In the present invention, the H chain V region derived from an antibody recognizes a cell surface molecule
(s) or intracellular molecule(s), for example, a protein (a receptor or a signal-transduction-related protein) or a sugar
chain of the protein or on cell membrane and oligomerizes, for example, dimerizes through crosslinking said molecule,
25 and thereby transduces a signal into the cells. The H chain V region of the invention includes H chain V regions derived
from a mammal (e.g. human, mouse, rat, bovine, sheep, ape etc.) and H chain V regions having partially modified
amino acid sequences of the H chain V regions. More preferable is a humanized H chain V region containing FR of H
chain V region of a human monoclonal antibody and CDR of H chain V region of a mouse monoclonal antibody. Also
preferable is an H chain V region having an amino acid sequence derived from a human, which can be produced by
30 recombination technique. The H chain V region of the invention may be a fragment of aforementioned H chain V region,
which fragment preserves the antigen binding capacity.
L chain V region
35 [0051] In the present invention, the L chain V region recognizes a cell surface molecule(s) or intracellular molecule
(s), for example, a protein (a receptor or a signal-transduction-related protein) or a sugar chain of the protein or on cell
membrane and oligomerizes, for example, dimerizes through crosslinking said molecule, and thereby transduces a
signal into the cells. The L chain V region of the invention includes L chain V regions derived from a mammal (e.g.
human, mouse, rat, bovine, sheep, ape etc.) and L chain V regions having partially modified amino acid sequences of
40 the L chain V regions. More preferable is a humanized L chain V region containing FR of L chain V region of human
monoclonal antibody and CDR of L chain V region of mouse monoclonal antibodies. Also preferable is an L chain V
region having an amino acid sequence derived from a human antibody, which can be produced by recombination
technique. The L chain V regions of the invention may be fragments of L chain V region, which fragments preserve
the antigen binding capacity.
45
Complementarity determining region (CDR)
[0052] Each V region of L chain and H chain forms an antigen-binding site. The variable region of the L and H chains
is composed of comparatively conserved four common framework regions linked to three hypervariable regions or
so complementarity determining regions (CDR) (Kabat, E.A. et al., "Sequences of Protein of Immunological Interest", US
Dept. Health and Human Services, 1983).
[0053] Major portions in the four framework regions (FRs) form (3-sheet structures and thus three CDRs form a loop.
CDRs may form a part of the p-sheet structure in certain cases. The three CDRs are held sterically close position to
each other by FR, which contributes to the formation of the antigen-binding site together with three CDRs.
55 [0054] These CDRs can be identified by comparing the amino acid sequence of V region of the obtained antibody
with known amino acid sequences of V regions of known antibodies according to the empirical rule in Kabat, E.A. et
al., "Sequences of Protein of Immunological Interest".
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Single chain Fv
[0055] A single chain Fv is a polypeptide monomer comprising an H chain V region and an L chain V region linked
each other which are derived from monoclonal antibodies. The resulting single chain Fvs contain variable regions of
s the parent monoclonal antibodies and preserve the complementarity determining region thereof, and therefore the
single chain Fvs bind to the antigen by the same specificity as that of the parent monoclonal antibodies (JP-Appl.
1 1 -63557). A part of the variable region and/or CDR of the single chain Fv of the invention or a part of the amino acid
sequence thereof may be partially altered, for example, deleted, replaced or added. The H chain V region and L chain
V region composing the single chain Fv of the invention are mentioned before and may be linked directly or through a
10 linker, preferably a peptide linker. The constitution of the single chain Fv may be [H chain V region]-[L chain V region]
or [L chain V region]-[H chain V region]. In the present invention, it is possible to make the single chain Fv to form a
dimer, a trimer or a tetramer, from which the modified antibody of the invention can be formed.
Single chain modified antibody
15
[0056] The single chain modified antibodies of the present invention comprising two or more H chain V regions and
two or more L chain V regions, preferably each two to four, especially preferable each two, comprise two or more H
chain V regions and L chain V regions as mentioned above. Each region of the peptide should be arranged such that
the modified single chain antibody forms a specific steric structure, concretely mimicking a steric structure formed by
20 the dimer of single chain Fv. For instance, the V regions are arranged in the order of the following manner:
[H chain V region]-[L chain V region]-[H chain V region]-[L chain V region]; or
[L chain V region]-[H chain V region]-[L chain V region]-[H chain V region],
25 wherein these regions are connected through a peptide linker, respectively.
Linker
[0057] In this invention, the linkers for the connection between the H chain V region and the L chain V region may
30 be any peptide linker which can be introduced by the genetic engineering procedure or any linker chemically synthe-
sized. For instance, linkers disclosed in literatures, e.g. Protein Engineering, 9(3), 299-305, 1996 may be used in the
invention. These linkers can be the same or different in the same molecule. If peptide linkers are required, the following
are cited as example linkers:
35 Ser
Gly-Ser
Gly-Gly-Ser
Ser-Gly-Gly
Gly-Gly-Gly-Ser
40 Ser-Gly-Gly-Gly
Gly-Gly-Gly-Gly-Ser
Ser-Gly-Gly-Gly-Gly
Gly-Gly-Gly-Gly-Gly-Ser
Ser-Gly-Gly-Gly-Gly-Gly
45 Gly-Gly-Gly-Gly-Gly-Gly-Ser
Ser-Gly-Gly-Gly-Gly-Gly-Gly
(Gly-Gly-Gly-Gly-Ser) n and
(Ser-Gly-Gly-Gly-Gly) n
so wherein n is an integer not less than one. Preferable length of the linker peptide varies dependent upon the receptor
to be the antigen, in the case of single chain Fvs, the range of 1 to 20 amino acids is normally preferable. In the case
of single chain modified antibodies comprising two or more H chain V regions and two or more L chain V regions, the
peptide linkers connecting those forming the same antigen binding site comprising [H chain V region]-[L chain V region]
(or [L chain V region]-[H chain V region]) have lengths of 1 - 30 amino acids, preferably 1 - 20 amino acids, more
55 preferably 3 - 1 8 amino acids. The peptide linkers connecting those not forming the same antigen biding site comprising
[H chain V region]-[L chain V region] or ([L chain V region]-[H chain V region]) have lengths of 1 - 40 amino acids,
preferably 3 - 30 amino acids, more preferably 5 - 20 amino acids. The method for introducing those linkers will be
described in the explanation for DNA construction coding for modified antibodies of the invention.
7
EP 1 327 681 A1
[0058] The chemically synthesized linkers, i.e. the chemical crosslinking agents, according to the invention can be
any linkers conventionally employed for the linkage of peptides. Examples of the linkers may include N-hydroxy suc-
cinimide (NHS), disuccinimidyl suberate (DSS), bis (sulfosuccinimidyl) suberate (BS 3 ), dithiobis (succinimidyl propi-
onate) (DSP), dithiobis(sulfosuccinimidyl propionate) (DTSSP), ethylene glycolbis(succinimidyl succinate) (EGS), eth-
s ylene glycolbis(sulfosuccinimidyl succinate) (sulfo-EGS), disuccinimidyl tartrate (DST), disulfosuccinimidyl tartrate (sul-
fo-DST), bis[2-(succinimido oxycarbonyloxy)ethyl]sulfone (BSOCOES), bis[2-(sulfosuccinimido oxycarbonyloxy) ethyl]
sulfone (sulfo-BSOCOES) or the like. These are commercially available. It is preferable for the chemically synthesized
linkers to have the length equivalent to that of peptide linkers.
[0059] To form a dimer of the single chain Fv it is preferable to select a linker suitable to dimerize in the solution such
10 as culture medium more than 20%, preferably more than 50%, more preferably more than 80%, most preferably more
than 90% of the single chain Fv produced in the host cells. Specifically, preferable is a linker composed of 2 to 12
amino acids, preferably 3 to 1 0 amino acids or other linkers corresponding thereto.
Preparation of modified antibodies
15
[0060] The modified antibodies can be produced by connecting, through the aforementioned linker, an H chain V
region and an L chain V region derived from known or novel monoclonal antibodies specifically binding to a cell surface
molecule(s). As examples of the single chain Fvs are cited MABL1 -scFv and MABL2-scFv comprising the H chain V
region and the L chain V region derived from the antibody MABL-1 and the antibody MABL-2, respectively. As examples
20 of the single chain polypeptides comprising two H chain V regions and two L chain V regions are cited MABL1 -sc(Fv) 2
and MABL2-sc(Fv) 2 comprising the H chain V region and the L chain V region derived from the aforementioned anti-
bodies.
[0061] For the preparation of the polypeptide, a signal peptide may be attached to N-terminal of the polypeptide if
the polypeptide is desired to be a secretory peptide. A well-known amino acid sequence useful for the purification of
25 polypeptide such as the FLAG sequence may be attached for the efficient purification of the polypeptide. In this case
a dimer can be formed by using anti-FLAG antibody.
[0062] For the preparation of the modified antibody of the invention, it is necessary to obtain a DNA, i.e. a DNA
encoding the single chain Fv or a DNA encoding reconstructed single chain polypeptide. These DNAs, especially for
MABL1-scFv, MABL2-scFv, MABL1 -sc(Fv) 2 and/or MABL2-SC(Fv) 2 are obtainable from the DNAs encoding the H
30 chain V region and the L chain V region derived from said Fv. They are also obtainable by polymerase chain reaction
(PCR) method using those DNA as a template and amplifying the part of DNA contained therein encoding desired
amino acid sequence with the aid of a pair of primers corresponding to both ends thereof.
[0063] In the case where each V region having partially modified amino acid sequence is desired, the V regions in
which one or some amino acids are modified, i.e. deleted, replaced or added can be obtained by a procedure known
35 in the art using PCR. A part of the amino acid sequence in the V region is preferably modified by the PCR known in
the art in order to prepare the modified antibody which is sufficiently active against the specific antigen.
[0064] For the determination of primers for the PCR amplification, it is necessary to decide the type of the H chain
and L chain of the desired antibodies. In the case of antibody MABL-1 and the antibody MABL-2 it has been reported,
however, that the antibody MABL-1 has k type L chains and y1 type H chains and the antibody MABL-2 has k type L
40 chains and 72a type H chains (JP-Appl. 11-63557). For the PCR amplification of the DNA encoding the H chain and L
chain of the antibody MABL-1 and/or the antibody MABL-2, primers described in Jones, ST. et al., Bio/Technology, 9,
88-89, 1991 may be employed.
[0065] For the amplification of the L chain V regions of the antibody MABL-1 and the antibody MABL-2 by PCR, 5'-
end and 3'-end oligonucleotide primers are decided as aforementioned. In the same manner, 5'-end and 3'-end oligo-
45 nucleotide primers are decided for the amplification of the H chain V regions of the antibody MABL-1 and the antibody
MABL-2.
[0066] In embodiments of the invention, the 5'-end primers which contain a sequence "GANTC" providing the re-
striction enzyme Hinf I recognition site at the neighborhood of 5'-terminal thereof are used and the 3'-end primers which
contain a nucleotide sequence "CCCGGG" providing the Xmal recognition site at the neighborhood of 5'-terminal there-
50 of are used. Other restriction enzyme recognition site may be used instead of these sites as long as they are used for
subcloning a desired DNA fragment into a cloning vector.
[0067] Specifically designed PCR primers are employed to provide suitable nucleotide sequences at 5'-end and 3'-
end of the cDNAs encoding the V regions of the antibodies MABL-1 and MABL-2 so that the cDNAs are readily inserted
into an expression vector and appropriately function in the expression vector (e.g. this invention devises to increase
55 translation efficiency by inserting Kozak sequence). The V regions of the antibodies MABL-1 and MABL-2 obtained by
amplifying by PCR using these primers are inserted into HEF expression vector containing the desired human C region
(see W092/19759). The cloned DNAs can be sequenced by using any conventional process, for example, by the
automatic DNA sequencer (Applied Biosystems).
8
EP 1 327 681 A1
[0068] A linker such as a peptide linker can be introduced into the modified antibody of the invention in the following
manner. Primers which have partially complementary sequence with the primers for the H chain V regions and the L
chain V regions as described above and which code for the N-terminal or the C-terminal of the linker are designed.
Then, the PCR procedure can be carried out using these primers to prepare a DNA encoding the peptide linker having
s desired amino acid sequence and length. The DNAs encoding the H chain V region and the L chain V region can be
connected through the resulting DNA to produce the DNA encoding the modified antibody of the invention which has
the desired peptide linker. Once the DNA encoding one of the modified antibodies is prepared, the DNAs encoding the
modified antibodies with or without the desired peptide linker can readily be produced by designing various primers
for the linker and then carrying out the PCR using the primers and the aforementioned DNA as a template.
10 [0069] Each V region of the modified antibody of the present invention can be humanized by using conventional
techniques (e.g. Sato, K. et al., Cancer Res., 53, 1 -6 (1 993)). Once a DNA encoding each of humanized Fvs is prepared,
a humanized single chain Fv, a fragment of the humanized single chain Fv, a humanized monoclonal antibody and a
fragment of the humanized monoclonal antibody can readily be produced according to conventional methods. Prefer-
ably, amino acid sequences of the V regions thereof may be partially modified, if necessary.
15 [0070] Furthermore, a DNA derived from other mammalian origin, for example a DNA encoding each of V regions
of human antibody, can be produced in the same manner as used to produce DNA encoding the H chain V region and
the L chain V region derived from mouse by conventional methods as mentioned in the above. The resulting DNA can
be used to prepare an H chain V region and an L chain V region of other mammal, especially derived from human
antibody, a single chain Fv derived from human and a fragment thereof, and a monoclonal antibody of human origin
20 and a fragment thereof.
[0071] When the modified antibodies of the invention is bi-specific modified antibodies, they can be produced by
known methods (for example, the method described in WO9413804).
[0072] As mentioned above, when the aimed DNAs encoding the V regions of the modified antibodies and the V
regions of the humanized modified antibodies are prepared, the expression vectors containing them and hosts trans-
25 formed with the vectors can be obtained according to conventional methods. Further, the hosts can be cultured ac-
cording to a conventional method to produce the reconstructed single chain Fv, the reconstructed humanized single
chain Fv, the humanized monoclonal antibodies and fragments thereof. They can be isolated from cells or a medium
and can be purified into a homogeneous mass. For this purpose any isolation and purification methods conventionally
used for proteins, e.g. chromatography, ultra-filtration, salting-out and dialysis, may be employed in combination, if
30 necessary, without limitation thereto.
[0073] When the reconstructed single chain Fv of the present invention is produced by culturing an animal cell such
as COS7 cells or CHO cells, preferably CHO cells, in a serum-free medium, the dimer of said single chain Fv formed
in the medium can be stably recovered and purified in a high yield. Thus purified dimer can be stably preserved for a
long period. The serum-free medium employed in the invention may be any medium conventionally used for the pro-
35 duction of a recombinant protein without limit thereto.
[0074] For the production of the modified antibodies of the present invention, any expression systems can be em-
ployed, for example, eukaryotic cells such as animal cells, e.g., established mammalian cell lines, filamentous fungi
and yeast, and prokaryotic cells such as bacterial cells e.g., E. coli. Preferably, the modified antibodies of the invention
are expressed in mammalian cells, for example COS7 cells or CHO cells.
40 [0075] In these cases, conventional promoters useful for the expression in mammalian cells can be used. Preferably,
human cytomegalovirus (HCMV) immediate early promoter is used. Expression vectors containing the HCMV promoter
include HCMV-VH-HCy 1 , HCMV-VL-HCK and the like which are derived from pSV2neo (W092/1 9759).
[0076] Additionally, other promoters for gene expression in mammal cell which may be used in the invention include
virus promoters derived form retrovirus, polyoma virus, adenovirus and simian virus 40 (SV40) and promoters derived
45 from mammal such as human polypeptide-chain elongation factor-1a (HEF-1a). SV40 promoter can easily be used
according to the method of Mulligan, R.C., et al. (Nature 277, 108-114 (1 979)) and HEF-1oc promoter can also be used
according to the methods of Mizushima, S. et al. (Nucleic Acids Research, 1 8, 5322 (1 990)).
[0077] Replication origin (ori) which can be used in the invention includes ori derived from SV40, polyoma virus,
adenovirus, bovine papilloma virus (BPV) and the like. An expression vector may contain, as a selection marker, phos-
50 photransferase APH (3') II or I (neo) gene, thymidine kinase (TK) gene, E. coli xanthine-guanine phosphoribosyl trans-
ferase (Ecogpt) gene or dihydrofolate reductase (DHFR) gene.
[0078] The antigen-binding activity of the modified antibody prepared in the above can be evaluated by a conventional
method such as radio immunoassay (RIA), enzyme-linked immunosorbent assay (ELISA) or surface plasmon reso-
nance. It can also be evaluated using the binding-inhibitory ability of original antibodies as an index, for example in
55 terms of the absence or presence of concentration-dependent inhibition of the binding of said monoclonal antibody to
the antigen.
[0079] More in detail, animal cells transformed with an expression vector containing a DNA encoding the modified
antibody of the invention, e.g., COS7 cells or CHO cells, are cultured. The cultured cells and/or the supernatant of the
9
EP 1 327 681 A1
medium or the modified antibody purified from them are used to determine the binding to antigen. As a control is used
a supernatant of the culture medium in which cells transformed only with the expression vector were cultured. In the
case of an antigen, for example, the antibody MABL-1 and the antibody MABL-2, a test sample of the modified antibody
of the invention or the supernatant of the control is added to mouse leukemia cell line, L1 21 0 cells, expressing human
s IAP and then an assay such as the flow cytometry is carried out to evaluate the antigen-binding activity.
[0080] In vitro evaluation of the signal transduction effect (apoptosis-inducing effect in the cases of the antibody
MABL-1 and the antibody MABL-2) is performed in the following manner: A test sample of the above modified antibody
is added to the cells which are expressing the antibody or cells into which the gene for the antibody has been introduced,
and is evaluated by the change caused by the signal transduction, for example, whether cell death is induced in a
10 manner specific to the human lAP-antigen, using conventional methods.
[0081] In vivo evaluation of the apoptosis-inducing effect, for example, in the case where the modified antibody
recognizes human IAP (e.g. modified antibodies derived from the antibody MABL-1 and the antibody MABL-2) is carried
out in the following manner: A mouse model of human myeloma is prepared. To the mice is intravenously administered
the monoclonal antibody or the modified antibody of the invention, which induces apoptosis of nucleated blood cells
15 having IAP. To mice of a control group is administered PBS alone. The induction of apoptosis is evaluated in terms of
antitumor effect based on the change of human IgG content in serum of the mice and their survival time.
[0082] As mentioned above the modified antibodies of the invention can be obtained by preparing modified antibodies
which contain two or more H chain V regions and two or more L chain V regions and specifically bind to target cell
surface molecule or intracellular molecule and screening the modified antibodies by in vivo or in vitro evaluation as
20 mentioned in the above.
[0083] The modified antibodies of the invention, which comprises two or more H chain V regions and two or more L
chain V regions, preferably each two to four, more preferably each two, may be a dimer of the single chain Fv comprising
one H chain V region and one L chain V region, or a single chain polypeptide in which two or more H chain V regions
and two or more L chain V regions are connected. It is considered that owing to such construction the peptide mimics
25 three dimensional structure of a natural ligand and therefore retains an excellent antigen-binding property and agonist
activity.
[0084] The modified antibodies of the invention have a remarkably lowered molecular size compared with antibody
molecule (whole IgG), and, therefore, a superior permeability into tissues and tumors and a higher activity than original
agonist monoclonal antibodies. Therefore, proper selection of the parent antibody makes it possible to transduce var-
30 ious signals into cells and to induce various actions in the cells such as apoptosis induction, cell proliferation induction,
cell differentiation induction, cell division induction or cell cycle regulation action. The pharmaceutical preparations
containing them are useful for treating diseases curable by inducing signal transduction, for example cancers, inflam-
mation, hormone disorders, autoimmune diseases as well as blood dyscrasia, for example, leukemia, malignant lym-
phoma, aplastic anemia, myelodysplasia syndrome and polycythemia vera. It is further expected that the antibody of
35 the invention can be used as a contrast agent by Rl-labeling. The effect can be enhanced by attaching to a Rl-compound
or a toxin.
BEST MODE FOR WORKING THE INVENTION
40 [0085] The present invention will concretely be illustrated in reference to the following examples, which in no way
limit the scope of the invention.
[0086] For illustrating the production process of the modified antibodies of the invention, examples of producing
single chain Fvs are shown below. Mouse antibodies against human IAP, MABL-1 and MABL-2 were used in the ex-
amples of producing the modified antibodies. Hybridomas MABL-1 and MABL-2 producing them respectively were
45 internationally deposited as FERM BP-6100 and FERM BP-6101 with the National Institute of Bioscience and Human
Technology, Agency of Industrial Science and Technology, Minister of International Trade and Industry (1-3 Higasi
1-chome, Tsukuba-shi, Ibaraki-ken, Japan), an authorized depository for microorganisms, on September 11, 1997.
Examples
50
Example 1 (Cloning of DNAs encoding V region of mouse monoclonal antibodies to human IAP)
[0087] DNAs encoding variable regions of the mouse monoclonal antibodies to human IAP, MABL-1 and MABL-2,
were cloned as follows.
55
1.1 Preparation of messenger RNA (mRNA)
[0088] mRNAs of the hybridomas MABL-1 and MABL-2 were obtained by using mRNA Purification Kit (Pharmacia
10
EP 1 327 681 A1
Biotech).
1 .2 Synthesis of double-stranded cDNA
s [0089] Double-stranded cDNA was synthesized from about 1 ng of the mRNA using Marathon cDNA Amplification
Kit (CLONTECH) and an adapter was linked thereto.
1 .3 PCR Amplification of genes encoding variable regions of an antibody by
10 [0090] PCR was carried out using Thermal Cycler (PERKIN ELMER).
(1 ) Amplification of a gene coding for L chain V region of MABL-1
[0091] Primers used for the PCR method are Adapter Primer-1 (CLONTECH) shown in SEQ ID No. 1 , which hybrid-
15 izes to a partial sequence of the adapter, and MKC (Mouse Kappa Constant) primer (Bio/Technology, 9, 88-89, 1 991 )
shown in SEQ ID No. 2, which hybridizes to the mouse kappa type L chain V region.
[0092] 50 \i\ of the PCR solution contains 5 nl of 10 X PCR Buffer II, 2 mM MgCI 2 , 0.16 mM dNTPs (dATP, dGTP,
dCTP and dTTP), 2.5 units of a DNA polymerase, AmpliTaq Gold (PERKIN ELMER), 0.2 ^M of the adapter primer of
SEQ ID No. 1, 0.2 iiM of the MKC primer of SEQ ID No. 2 and 0.1 |ig of the double-stranded cDNA derived from MABL-
20 1. The solution was preheated at 94°C of the initial temperature for 9 minutes and then heated at 94°C for 1 minute,
at 60°C for 1 minute and at 72°C for 1 minute 20 seconds in order. This temperature cycle was repeated 35 times and
then the reaction mixture was further heated at 72°C for 10 minutes.
(2) Amplification of cDNA encoding H chain V region of MABL-1
25
[0093] The Adapter Primer-1 shown in SEQ ID No. 1 and MHC-y1 (Mouse Heavy Constant) primer (Bio/Technology,
9, 88-89, 1 991 ) shown in SEQ ID No. 3 were used as primers for PCR.
[0094] The amplification of cDNA was performed according to the method of the amplification of the L chain V region
gene, which was described in Example 1 .3-(1 ), except for using 0.2 |^M of the MHC-y1 primer instead of 0.2 |^M of the
30 MKC primer.
(3) Amplification of cDNA encoding L chain V region of MABL-2
[0095] The Adapter Primer-1 of SEQ ID No. 1 and the MKC primer of SEQ ID No. 2 were used as primers for PCR.
35 [0096] The amplification of cDNA was carried out according to the method of the amplification of the L chain V region
gene of MABL-1 which was described in Example 1.3-(1), except for using 0.1 [ig of the double-stranded cDNA derived
from MABL-2 instead of 0.1 ng of the double-stranded cDNA from MABL-1 .
(4) Amplification of cDNA encoding H chain V region of MABL-2
40
[0097] The Adapter Primer-1 of SEQ ID No. 1 and MHC-y2a primer (Bio/Technology, 9, 88-89, 1 991 ) shown in SEQ
ID No. 4 were used as primers for PCR.
[0098] The amplification of cDNA was performed according to the method of the amplification of the L chain V region
gene, which was described in Example 1 .3-(3), except for using 0.2 |xM of the MHC-y2a primer instead of 0.2 ^M of
45 the MKC primer.
1.4 Purification of PCR products
[0099] The DNA fragment amplified by PCR as described above was purified using the QIAquick PCR Purification
so Kit (QIAGEN) and dissolved in 10 mM Tris-HCI (pH 8.0) containing 1 mM EDTA.
1 .5 Ligation and Transformation
[0100] About 140 ng of the DNA fragment comprising the gene encoding the mouse kappa type L chain V region
55 derived from MABL-1 as prepared above was ligated with 50 ng of pGEM-T Easy vector (Promega) in the reaction
buffer comprising 30 mM Tris-HCI (pH 7.8), 10 mM MgCI 2 , 10 mM dithiothreitol, 1 mM ATP and 3 units of T4 DNA
Ligase (Promega) at 15°C for 3 hours.
[0101] Then, 1 |^l of the reaction mixture was added to 50 |^l of E^ coli DH5oc competent cells (Toyobo Inc.) and the
11
EP 1 327 681 A1
cells were stored on ice for 30 minutes, incubated at 42°C for 1 minute and stored on ice for 2 minutes again. 100 |^l
of SOC medium (GIBCO BRL) was added. The cells of coli were plated on LB (Molecular Cloning: A Laboratory
Manual, Sambrook et al., Cold Spring Harbor Laboratory Press, 1 989) agar medium containing 1 00 ng/ml of ampicillin
(SIGMA) and cultured at 37°C overnight to obtain the transformant of E. coli.
s [0102] The transformant was cultured in 3 ml of LB medium containing 50 |^g/ml of ampicillin at 37°C overnight and
the plasmid DNA was prepared from the culture using the QIAprep Spin Miniprep Kit (QIAGEN).
[0103] The resulting plasmid comprising the gene encoding the mouse kappa type L chain V region derived from
the hybridoma MABL-1 was designated as pGEM-M1L.
[0104] According to the same manner as described above, a plasmid comprising the gene encoding the mouse H
10 chain V region derived from the hybridoma MABL-1 was prepared from the purified DNA fragment and designated as
pGEM-M1H.
[0105] A plasmid comprising the gene encoding the mouse kappa type L chain V region derived from the hybridoma
MABL-2 was prepared from the purified DNA fragment and designated as pGEM-M2L.
[0106] A plasmid comprising the gene encoding the mouse H chain V region derived from the hybridoma MABL-2
15 was prepared from the purified DNA fragment and designated as pGEM-M2H.
Example 2 (DNA Sequencing)
[0107] The nucleotide sequence of the cDNA encoding region in the aforementioned plasmids was determined using
20 Auto DNA Sequencer (Applied Biosystem) and ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction Kit
(Applied Biosystem) according to the manufacturer's protocol.
[0108] The nucleotide sequence of the gene encoding the L chain V region from the mouse antibody MABL-1 , which
is included in the plasmid pGEM-M1L, is shown in SEQ ID No. 5.
[0109] The nucleotide sequence of the gene encoding the H chain V region from the mouse antibody MABL-1 , which
25 is included in the plasmid pGEM-M1 H, is shown in SEQ ID No. 6.
[01 10] The nucleotide sequence of the gene encoding the L chain V region from the mouse antibody MABL-2, which
is included in the plasmid pGEM-M2L, is shown in SEQ ID No. 7.
[01 1 1] The nucleotide sequence of the gene encoding the H chain V region from the mouse antibody MABL-2, which
is included in the plasmid pGEM-M2H, is shown in SEQ ID No. 8.
30
Example 3 (Determination of CDR)
[01 12] The V regions of L chain and H chain generally have a similarity in their structures and each four framework
regions therein are linked by three hypervariable regions, i.e., complementarity determining regions (CDR). An amino
35 acid sequence of the framework is relatively well conserved, while an amino acid sequence of CDR has extremely high
variation (Kabat, E.A., et al., "Sequences of Proteins of Immunological Interest", US Dept. Health and Human Services,
1983).
[0113] On the basis of these facts, the amino acid sequences of the variable regions from the mouse monoclonal
antibodies to human IAP were applied to the database of amino acid sequences of the antibodies made by Kabat et
40 al. to investigate the homology. The CDR regions were determined based on the homology as shown in Table 1 .
Table 1
Plasmid
SEQ ID No.
CDR(1)
CDR(2)
CDR(3)
pGEM-M1 L
5
43-58
74-80
113-121
pGEM-M1H
6
50-54
69-85
118-125
pGEM-M2L
7
43-58
74-80
113-121
pGEM-M2H
8
50-54
69-85
118-125
50
55
12
EP 1 327 681 A1
Example 4 (Identification of Cloned cDNA Expression
(Preparation of Chimera MABL-1 antibody and Chimera MABL-2 antibody.)
s 4.1 Preparation of vectors expressing chimera MABL-1
antibody
[0114] cDNA clones, pGEM-M1L and pGEM-M1 H, encoding the V regions of the L chain and the H chain of the
10 mouse antibody MABL-1, respectively, were modified by the PCR method and introduced into the HEF expression
vector (W092/19759) to prepare vectors expressing chimera MABL-1 antibody.
[0115] A forward primer MLS (SEQ ID No. 9) for the L chain V region and a forward primer MHS (SEQ ID No. 10)
for the H chain V region were designed to hybridize to a DNA encoding the beginning of the leader sequence of each
V region and to contain the Kozak consensus sequence (J. Mol. Biol., 196, 947-950, 1987) and Hind 1 1 1 restriction
15 enzyme site. A reverse primer MLAS (SEQ ID No. 11) for the L chain V region and a reverse primer MHAS (SEQ ID
No. 12) for the H chain V region were designed to hybridize to a DNA encoding the end of the J region and to contain
the splice donor sequence and BamHI restriction enzyme site.
[0116] 100 \i\ of a PCR solution comprising 10 \i\ of 10 x PCR Buffer II, 2 mM MgCI 2 , 0.16 mM dNTPs (dATP, dGTP,
dCTP and dTTP), 5 units of DNA polymerase AmpliTaq Gold, 0.4 |^M each of primers and 8 ng of the template DNA
20 (pGEM-M1 L or pGEM-M1 H) was preheated at 94°C of the initial temperature for 9 minutes and then heated at 94°C
for 1 minute, at 60°C for 1 minute and at 72°C for 1 minute 20 seconds in order. This temperature cycle was repeated
35 times and then the reaction mixture was further heated at 72°C for 10 minutes.
[0117] The PCR product was purified using the QIAquick PCR Purification Kit (QIAGEN) and then digested with
Hindlll and BamHI. The product from the L chain V region was cloned into the HEF expression vector, HEF-k and the
25 product from the H chain V region was cloned into the HEF expression vector, HEF-y. After DNA sequencing, plasmids
containing a DNA fragment with a correct DNA sequence are designated as HEF-M1 L and HEF-M1 H, respectively.
4.2 Preparation of vectors expressing chimera MABL-2 antibodies
30 [0118] Modification and cloning of cDNA were performed in the same manner described in Example 4.1 except for
using pGEM-M2L and pGEM-M2H as template DNA instead of pGEM-M1 L and pGEM-M1 H. After DNA sequencing,
plasmids containing DNA fragments with correct DNA sequences are designated as HEF-M2L and HEF-M2H, respec-
tively.
35 4.3 Transfection to COS7 cells
[01 19] The aforementioned expression vectors were tested in COS7 cells to observe the transient expression of the
chimera MABL-1 and MABL-2 antibodies.
40 (1 ) Transfection with genes for the chimera MABL-1 antibody
[0120] COS7 cells were co-transformed with the HEF-M1 L and HEF-M1 H vectors by electroporation using the Gene
Pulser apparatus (BioRad). Each DNA (1 0 ng) and 0.8 ml of PBS with 1 x 1 0 7 cells/ml were added to a cuvette. The
mixture was treated with pulse at 1 .5 kV, 25 \\F of electric capacity.
45 [0121] After the restoration for 10 minutes at a room temperature, the electroporated cells were transferred into
DMEM culture medium (GIBCO BRL) containing 1 0% y-globulin-free fetal bovine serum. After culturing for 72 hours,
the supernatant was collected, centrifuged to remove cell fragments and recovered.
(2) Transfection with genes coding for the chimera MABL-2 antibody
50
[0122] The co-transfection to COS7 cells with the genes coding for the chimera MABL-2 antibody was carried out in
the same manner as described in Example 4.3-(1 ) except for using the HEF-M2L and HEF-M2H vectors instead of the
HEF-M1L and HEF-M1H vectors. The supernatant was recovered in the same manner.
55 4.4 Flow cytometry
[0123] Flow cytometry was performed using the aforementioned culture supernatant of COS7 cells to measure bind-
ing to the antigen. The culture supernatant of the COS7 cells expressing the chimera MABL-1 antibody or the COS7
13
EP 1 327 681 A1
cells expressing the chimera MABL-2 antibody, or human IgG antibody (SIGMA) as a control was added to 4 x 10 5
cells of mouse leukemia cell line L121 0 expressing human IAP and incubated on ice. After washing, the FITC-labeled
anti-human IgG antibody (Cappel) was added thereto. After incubating and washing, the fluorescence intensity thereof
was measured using the FACScan apparatus (BECTON DICKINSON).
s [0124] Since the chimera MABL-1 and MABL-2 antibodies were specifically bound to L1 21 0 cells expressing human
IAP, it is confirmed that these chimera antibodies have proper structures of the V regions of the mouse monoclonal
antibodies MABL-1 and MABL-2, respectively (Figs. 1-3).
Example 5 (Preparation of reconstructed Single chain Fv (scFv) of the antibody MABL-1 and antibody MABL-2)
10
5.1 Preparation of reconstructed single chain Fv of antibody MABL-1
[0125] The reconstructed single chain Fv of antibody MABL-1 was prepared as follows. The H chain V region and
the L chain V of antibody MABL-1 , and a linker were respectively amplified by the PCR method and were connected
15 to produce the reconstructed single chain Fv of antibody MABL-1. The production method is illustrated in Fig. 4. Six
primers (A-F) were employed for the production of the single chain Fv of antibody MABL-1 . Primers A, C and E have
a sense sequence and primers B, D and F have an antisense sequence.
[0126] The forward primer VHS for the H chain V region (Primer A, SEQ ID No. 13) was designed to hybridize to a
DNA encoding the N-terminal of the H chain V region and to contain Ncol restriction enzyme recognition site. The
20 reverse primer VHAS for H chain V region (Primer B, SEQ ID No. 14) was designed to hybridize to a DNA coding the
C-terminal of the H chain V region and to overlap with the linker.
[0127] The forward primer LS for the linker (Primer C, SEQ ID No. 15) was designed to hybridize to a DNA encoding
the N-terminal of the linker and to overlap with a DNA encoding the C-terminal of the H chain V region. The reverse
primer LAS for the linker (Primer D, SEQ ID No. 16) was designed to hybridize to a DNA encoding the C-terminal of
25 the linker and to overlap with a DNA encoding the N-terminal of the L chain V region.
[0128] The forward primer VLS for the L chain V region (Primer E, SEQ ID No. 1 7) was designed to hybridize to a
DNA encoding the C-terminal of the linker and to overlap with a DNA encoding the N-terminal of the L chain V region.
The reverse primer VLAS-FLAG for L chain V region (Primer F, SEQ ID No. 18) was designed to hybridize to a DNA
encoding the C-terminal of the L chain V region and to have a sequence encoding the FLAG peptide (Hopp. T. P. et
30 al., Bio/Technology, 6, 1204-1210, 1988), two stop codons and EcoRI restriction enzyme recognition site.
[0129] In the first PCR step, three reactions, A-B, C-D and E-F, were carried out and PCR products thereof were
purified. Three PCR products obtained from the first PCR step were assembled by their complementarity. Then, the
primers A and F were added and the full length DNA encoding the reconstructed single chain Fv of antibody MABL-1
was amplified (Second PCR). In the first PCR, the plasmid pGEM-M1H encoding the H chain V region of antibody
35 MABL-1 (see Example 2), a plasmid pSC-DP1 which comprises a DNA sequence encoding a linker region comprising:
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser (SEQ ID No. 19) (Huston, J.S., et al., Proc. Natl. Acad.
Sci. USA, 85, 5879-5883, 1 988) and the plasmid pGEM-M1 L encoding the L chain V region of antibody MABL-1 (see
Example 2) were employed as template, respectively.
[0130] 50 |xl of the solution for the first PCR step comprises 5^1 of 10 x PCR Buffer II, 2 mM MgCI 2 , 0.16 mM dNTPs,
40 2.5 units of DNA polymerase, AmpliTaq Gold (PERKIN ELMER), 0.4 |^M each of primers and 5 ng each of template
DNA. The PCR solution was preheated at 94°C of the initial temperature for 9 minutes and then heated at 94°C for 1
minute, at 65°C for 1 minute and at 72°C for 1 minute and 20 seconds in order. This temperature cycle was repeated
35 times and then the reaction mixture was further heated at 72°C for 7 minutes.
[0131] The PCR products A-B (371 bp), C-D (63bp) and E-F (384bp) were purified using the QIAquick PCR Purifi-
es cation Kit (QIAGEN) and were assembled in the second PCR. In the second PCR, 98 of a PCR solution comprising
120 ng of the first PCR product A-B, 20 ng of the PCR product C-D and 120 ng of the PCR product E-F, 10 (il of 10 x
PCR Buffer II, 2mM MgCI 2 , 0.16 mM dNTPs, 5 units of DNA polymerase AmpliTaq Gold (PERKIN ELMER) was pre-
heated at 94°C of the initial temperature for 8 minutes and then heated at 94°C for 2 minutes, at 65°C for 2 minutes
and at 72°C for 2 minutes in order. This temperature cycle was repeated twice and then 0.4 jaM each of primers A and
so F were added into the reaction, respectively. The mixture was preheated at 94°C of the initial temperature for 1 minutes
and then heated at 94°C for 1 minute, at 65°C for 1 minute and at 72°C for 1 minute and 20 seconds in order. This
temperature cycle was repeated 35 times and then the reaction mixture was further heated at 72°C for 7 minutes.
[0132] A DNA fragment of 843 bp produced by the second PCR was purified and digested by Ncol and EcoRI. The
resultant DNA fragment was cloned into pSCFVT7 vector. The expression vector pSCFVT7 contains a pelB signal
55 sequence suitable for E^ coli periplasmic expression system (Lei, S.P., et al., J. Bacteriology, 1 69, 4379-4383, 1 987).
After the DNA sequencing, the plasmid containing the DNA fragment encoding correct amino acid sequence of the
reconstructed single chain Fv of antibody MABL-1 is designated as "pscM1" (see Fig. 5). The nucleotide sequence
and the amino acid sequence of the reconstructed single chain Fv of antibody MABL-1 contained in the plasmid pscM1
14
EP 1 327 681 A1
are shown in SEQ ID No. 20.
[0133] The pscM1 vector was modified by the PCR method to prepare a vector expressing the reconstructed single
chain Fv of antibody MABL-1 in mammalian cells. The resultant DNA fragment was introduced into pCHOI expression
vector. This expression vector, pCHOI , was constructed by digesting DHFR-AE-rvH-PM1 -f (W092/1 9759 with EcoRI
s and Smal to eliminate the antibody gene and connecting the EcoRI-Notl-BamHI Adapter (Takara Shuzo) thereto.
[0134] As a forward primer for PCR, Sal-VHS primer shown in SEQ ID No. 21 was designed to hybridize to a DNA
encoding the N-terminal of the H chain V region and to contain Sail restriction enzyme recognition site. As a reverse
primer for PCR, FRHIanti primer shown in SEQ ID No. 22 was designed to hybridize to a DNA encoding the end of
the first framework sequence.
10 [0135] 100 \i\ of PCR solution comprising 10 jllI of 10 x PCR Buffer II, 2 mM MgCI 2 , 0.16 mM dNTPs, 5 units of the
DNA polymerase, AmpliTaq Gold, 0.4 |il M each of primer and 8 ng of the template DNA (pscM1) was preheated at
95°C of the initial temperature for 9 minutes and then heated at 95°C for 1 minute, at 60°C for 1 minute and at 72°C
for 1 minute and 20 seconds in order. This temperature cycle was repeated 35 times and then the reaction mixture
was further heated at 72°C for 7 minutes.
15 [0136] The PCR product was purified using the QIAquick PCR Purification Kit (QIAGEN) and digested by Sail and
Mboll to obtain a DNA fragment encoding the N-terminal of the reconstructed single chain Fv of antibody MABL-1 The
pscM1 vector was digested by Mboll and EcoRI to obtain a DNA fragment encoding the C-terminal of the reconstructed
single chain Fv of antibody MABL-1 . The Sall-Mboll DNA fragment and the Mboll-EcoRI DNA fragment were cloned
into pCHOI -Igs vector. After DNA sequencing, the plasmid comprising the desired DNA sequence was designated as
20 "pCHOMI " (see Fig. 6). The expression vector, pCHOI -Igs, contains a mouse lgG1 signal sequence suitable for the
secretion-expression system in mammalian cells (Nature, 322, 323-327, 1988). The nucleotide sequence and the
amino acid sequence of the reconstructed single chain Fv of antibody MABL-1 contained in the plasmid pCHOMI are
shown in SEQ ID No. 23.
25 5.2 Preparation of reconstructed single chain Fv of antibody MABL-2
[0137] The reconstructed single chain Fv of antibody MABL-2 was prepared in accordance with the aforementioned
Example 5.1 . Employed in the first PCR step were plasmid pGEM-M2H encoding the H chain V region of MABL-2 (see
Example 2) instead of pGEM-M1H and plasmid pGEM-M2L encoding the L chain V region of MABL-2 (see Example
30 2) instead of pGEM-M1L, to obtain a plasmid pscM2 which comprises a DNA fragment encoding the desired amino
acid sequence of the single chain Fv of antibody MABL-2. The nucleotide sequence and the amino acid sequence of
the reconstructed single chain Fv of antibody MABL-2 contained in the plasmid pscM2 are shown in SEQ ID No. 24.
[0138] The pscM2 vector was modified by the PCR method to prepare a vector, pCHOM2, for the expression in
mammalian cells which contains the DNA fragment encoding the correct amino acid sequence of reconstructed the
35 single chain Fv of. antibody MABL-2. The nucleotide sequence and the amino acid sequence of the reconstructed
single chain Fv of antibody MABL-2 contained in the plasmid pCHOM2 are shown in SEQ ID No. 25.
5.3 Transfection to COS7 cells
40 [0139] The pCHOM2 vector was tested in COS7 cells to observe the transient expression of the reconstructed single
chain Fvof antibody MABL-2.
[0140] The COS7 cells were transformed with the pCHOM2 vector by electroporation using the Gene Pulser appa-
ratus (BioRad). The DNA (10 ng) and 0.8 ml of PBS with 1 x 10 7 cells/ml were added to a cuvette. The mixture was
treated with pulse at 1 .5 kV, 25 \jlF of electric capacity.
45 [0141] After the restoration for 10 minutes at a room temperature, the electroporated cells were transferred into
IMDM culture medium (GIBCO BRL) containing 1 0% fetal bovine serum. After culturing for 72 hours, the supernatant
was collected, centrifuged to remove cell fragments and recovered.
5.4 Detection of the reconstructed single chain Fv of antibody MABL-2 in culture supernatant of COS7 cells
50
[0142] The existence of the single chain Fv of antibody MABL-2 in the culture supernatant of COS7 cells which had
been transfected with the pCHOM2 vector was confirmed by the Western Blotting method.
[0143] The culture supernatant of COS7 cells transfected with the pCHOM2 vector and the culture supernatant of
COS7 cells transfected with the pCHOI as a control were subjected to SDS electrophoresis and transferred to REIN-
55 FORCED NC membrane (Schleicher & Schuell). The membrane was blocked with 5% skim milk (Morinaga Nyu-gyo),
washed with 0.05% Tween 20-PBS and mixed with an anti-FLAG antibody (SIGMA). The membrane was incubated
at room temperature, washed and mixed with alkaline phosphatase-conjugated mouse IgG antibody (Zymed). After
incubating and washing at room temperature, the substrate solution (Kirkegaard Perry Laboratories) was added to
15
EP 1 327 681 A1
develop color (Fig. 7).
[0144] A FLAG-peptide-specific protein was detected only in the culture supernatant of the pCHOM2 vector-intro-
duced COS7 cells and thus it is confirmed that the reconstructed single chain Fv of antibody MABL-2 was secreted in
this culture supernatant.
5
5.5 Flow cytometry
[0145] Flow cytometry was performed using the aforementioned COS7 cells culture supernatant to measure the
binding to the antigen. The culture supernatant of the COS7 cells expressing the reconstructed single chain Fv of
10 antibody MABL-2 or the culture supernatant of COS7 cells transformed with pCHOI vector as a control was added to
2 x 1 05 cells of the mouse leukemia cell line L1 21 0 expressing human Integrin Associated Protein (IAP) or the cell
line L1210 transformed with pCOS1 as a control. After incubating on ice and washing, the mouse anti-FLAG antibody
(SIGMA) was added. Then the cells were incubated and washed. Then, the FITC labeled anti-mouse IgG antibody
(BECTON DICKINSON) was added thereto and the cells were incubated and washed again. Subsequently, the fluo-
15 rescence intensity was measured using the FACScan apparatus (BECTON DICKINSON).
[0146] Since the single chain Fv of antibody MABL-2 was specifically bound to L1210 cells expressing human IAP,
it is confirmed that the reconstructed single chain Fv of antibody MABL-2 has an affinity to human Integrin Associated
Protein (IAP) (see Figs. 8-11).
20 5.6 Competitive ELISA
[0147] The binding activity of the reconstructed single chain Fv of antibody MABL-2 was measured based on the
inhibiting activity against the binding of mouse monoclonal antibodies to the antigen.
[0148] The anti-FLAG antibody adjusted to 1 ng/ml was added to each well on 96-well plate and incubated at37°C
25 for 2 hours. After washing, blocking was performed with 1% BSA-PBS. After incubating and washing at a room tem-
perature, the culture supernatant of COS7 cells into which the secretion-type human IAP antigen gene (SEQ ID No.
26) had been introduced was diluted with PBS into twofold volume and added to each well. After incubating and washing
at a room temperature, a mixture of 50 |^l of the biotinized MABL-2 antibody adjusted to 100 ng/ml and 50 |^l of se-
quentially diluted supernatant of the COS7 cells expressing the reconstructed single chain Fv of antibody MABL-2
30 were added into each well. After incubating and washing at a room temperature, the alkaline phosphatase-conjugated
streptoavidin (Zymed) was added into each well. After incubating and washing at a room temperature, the substrate
represent living cells and dots in the right-lower region represent cells at the early stage of apoptosis and dots in the
right-upper region represent cells at the late stage of apoptosis. The results show that the reconstructed single chain
Fv of antibody MABL-2 (MABL2-scFv) remarkably induced cell death of L1210 cells specific to human IAP antigen
35 (Figs. 13-16) and that the reconstructed single chain Fv also induced remarkable cell death of CCRF-CEM cells in
comparison with the control (Figs. 17-18).
5.8 Expression of MABL-2 derived single chain Fv in CHO cells
40 [0149] CHO cells were transfected with the pCHOM2 vector to establish a CHO cell line which constantly expresses
the single chain Fv (polypeptide) derived from the antibody MABL-2.
[0150] CHO cells were transformed with the pCHOM2 vector by the electroporation using the Gene Pulser apparatus
(BioRad). A mixture of DNA (1 0 jig) and 0.7 ml of PBS with CHO cells (1 x 1 0 7 cells/ml) was added to a cuvette. The
mixture was treated with pulse at 1.5 kV, 25 |xF of electric capacity. After the restoration for 10 minutes at a room
45 temperature, the electroporated cells were transferred into nucleic acid free oe-MEM medium (GIBCO BRL) containing
10% fetal bovine serum and cultured. The expression of desired protein in the resultant clones was confirmed by
SDS-PAGE and a clone with a high expression level was selected as a cell line producing the single chain Fv derived
from the solution (SIGMA) was added and absorbance of the reaction mixture in each well was measured at 405 nm.
[0151] The results revealed that the reconstructed single chain Fv of antibody MABL-2 (MABL2-scFv) evidently in-
50 hibited concentration-dependently the binding of the mouse antibody MABL-2 to human IAP antigen in comparison
with the culture supernatant of the PCHOI -introduced COS7 cells as a control (Fig. 12). Accordingly, it is suggested
that the reconstructed single chain Fv of antibody MABL-2 has the correct structure of each of the V regions from the
mouse monoclonal antibody MABL-2.
55 5.7 Apoptosis-inducing Effect in vitro
[0152] An apoptosis-inducing action of the reconstructed single chain Fv of antibody MABL-2 was examined by
Annexin-V staining (Boehringer Mannheim) using the L1210 cells transfected with human IAP gene, the L1210 cells
16
EP 1 327 681 A1
transfected with the pCOS1 vector as a control and CCRF-CEM cells.
[0153] To each 1 x 1 0 5 cells of the above cells was added the culture supernatant of the COS7 cells expressing the
reconstructed single chain Fv of antibody MABL-2 or the culture supernatant of COS7 cells transfected with the pCHOI
vector as a control at 50% final concentration and the mixtures were cultured for 24 hours. Then, the Annexin-V staining
s was performed and the fluorescence intensity was measured using the FACScan apparatus (BECTON DICKINSON).
[0154] Results of the Annexin-V staining are shown in Figs. 13-1 8, respectively. Dots in the left-lower region antibody
MABL-2. The cell line was cultured in serum-free medium CHO-S-SFM II (GIBCO BRL) containing 10 nM methotrexate
(SIGMA). Then, the culture supernatant was collected, centrifuged to remove cell fragments and recovered.
10 5.9 Purification of MABL-2 derived single chain Fv produced in CHO cells
[0155] The culture supernatant of the CHO cell line expressing the single chain Fv obtained in Example 5.8 was
concentrated up to twenty times using a cartridge for the artificial dialysis (PAN130SF, ASAHI MEDICALS). The con-
centrated solution was stored at -20°C and thawed on purification.
15 [0156] Purification of the single chain Fv from the culture supernatant of the CHO cells was performed using three
kinds of chromatography, i.e., Blue-sepharose, a hydroxyapatite and a gel filtration.
(1 ) Blue-sepharose column chromatography
20 [0157] The concentrated supernatant was diluted to ten times with 20 mM acetate buffer (pH 6.0) and centrifuged
to remove insoluble materials (10000 X rpm, 30 minutes). The supernatant was applied onto a Blue-sepharose column
(20 ml) equilibrated with the same buffer. After washing the column with the same buffer, proteins adsorbed in the
column were eluted by a stepwise gradient of NaCI in the same buffer, 0.1 , 0.2, 0.3, 0.5 and up to 1 .0 M. The pass-
through fraction and each eluted fraction were analyzed by SDS-PAGE. The fractions in which the single chain Fv were
25 confirmed (the fractions eluted at 0.1 to 0.3M NaCI) were pooled and concentrated up to approximately 20 times using
CentriPrep-10 (AMICON).
(2) Hydroxyapatite
30 [0158] The concentrated solution obtained in (1 ) was diluted to 1 0 times with 1 0 mM phosphate buffer (pH 7.0) and
applied onto the hydroxyapatite column (20 ml, BIORAD). The column was washed with 60 ml of 10 mM phosphate
buffer (pH 7.0). Then, proteins adsorbed in the column were eluted by a linear gradient of sodium phosphate buffer up
to 200 mM (see Fig. 19). The analysis of each fraction by SDS-PAGE confirmed the single chain Fv in fraction A and
fraction B.
35
(3) Gel filtration
[0159] Each of fractions A and B in (2) was separately concentrated with CentriPrep-1 0 and applied onto TSKgel
G3000SWG column (21.5 x 600 mm) equilibrated with 20 mM acetate buffer (pH 6.0) containing 0.15 M NaCI. Chro-
40 matograms are shown in Fig. 20. The analysis of the fractions by SDS-PAGE confirmed that both major peaks (Al and
Bl) are of desired single chain Fv. In the gel filtration analysis, the fraction A was eluted at 36 kDaof apparent molecular
weight and the fraction B was eluted at 76 kDa. The purified single chain Fvs (Al, Bl) were analyzed with 15% SDS
polyacrylamide gel. Samples were treated in the absence or presence of a reductant and the electrophoresis was
carried out in accordance with the Laemmli's method. Then the protein was stained with Coomassie Brilliant Blue. As
45 shown in Fig. 21 , both Al and Bl gave a single band at 35 kDa of apparent molecular weight, regardless of the absence
or presence of the reductant. From the above, it is concluded that Al is a monomer of the single chain Fv and Bl is a
non-covalently bound dimer of the single chain Fv. The gel filtration analysis of the fractions Al and Bl with TSKgel
G3000SW column (7.5 x 60 mm) revealed that a peak of the monomer is detected only in the fraction Al and a peak
of the dimer is detected only in the fraction Bl (Fig. 22). The dimer fraction (fraction Bl) accounted for 4 period of total
so single chain Fvs. More than 90% of the dimer in the dimer fraction was stably preserved for more than a month at 4°C.
5.1 0 Construction of vector expressing single chain Fv derived from antibody MABL-2 in E. coli cell
[0160] The pscM2 vector was modified by the PCR method to prepare a vector effectively expressing the single
55 chain Fvfrom the antibody MABL-2 in E^ coli cells. The resultant DNA fragment was introduced into pSCFVT7 expres-
sion vector.
[0161] As a forward primer for PCR, Nde-VHSm02 primer shown in SEQ ID No. 27 was designed to hybridize to a
DNA encoding the N-terminal of the H chain V region and to contain a start codon and Ndel restriction enzyme recog-
17
EP 1 327 681 A1
nition site. As a reverse primer for PCR, VLAS primer shown in SEQ ID No. 28 was designed to hybridize to a DNA
encoding the C-terminal of the L chain V region and to contain two stop codons and EcoRI restriction enzyme recognition
site. The forward primer, Nde-VHSm02, comprises five point mutations in the part hybridizing to the DNA encoding the
N-terminal of the H chain V region for the effective expression in E. coli.
s [0162] 100 nl of a PCR solution comprising 1 0 jxl of 1 0 x PCR Buffer #1,1 mM MgCI 2 , 0.2 mM dNTPs, 5 units of
KOD DNA polymerase (all from TOYOBO), 1 |xM of each primer and 100 ng of a template DNA (pscM2) was heated
at 98°C for 15 seconds, at 65°C for 2 seconds and at 74°C for 30 seconds in order. This temperature cycle was repeated
25 times.
[0163] The PCR product was purified using the QIAquick PCR Purification Kit (QIAGEN) and digested by Ndel and
10 EcoRI, and then the resulting DNA fragment was cloned into pSCFVT7 vector, from which pelB signal sequence had
been eliminated by the digestion with Ndel and EcoRI. After DNA sequencing, the resulting plasmid comprising a DNA
fragment with the desired DNA sequence is designated as "pscM2DEm02" (see Fig. 23). The nucleotide sequence
and the amino acid sequence of the single chain Fv derived from the antibody MABL-2 contained in the plasmid
pscM2DEm02 are shown in SEQ ID No. 29.
15
5.11 Expression of single chain Fv derived from antibody MABL-2 in E. coli cells
[0164] E. coli BL21(DE3)pLysS (STRATAGENE) was transformed with pscM2DEm02 vector to obtain a strain of E.
coli expressing the single chain Fv derived from antibody MABL-2. The resulting clones were examined for the expres-
20 sion of the desired protein using SDS-PAGE, and a clone with a high expression level was selected as a strain producing
the single chain Fv derived from antibody MABL-2.
5.12 Purification of single chain Fv derived from antibody MABL-2 produced in E.coli
25 [0165] A single colony of E^ coli obtained by the transformation was cultured in 3 ml of LB medium at 28°C for 7
hours and then in 70 ml of LB medium at 28°C overnight. This pre-culture was transplanted to 7 L of LB medium and
cultured at 28°C with stirring at 300 rpm using the Jarfermenter. When an absorbance of the medium reached O.D.
=1 .5, the bacteria were induced with 1 mM IPTG and then cultured for 3 hours.
[0166] The culture medium was centrifuged (10000 x g, 10 minutes) and the precipitated bacteria were recovered.
30 To the bacteria was added 50 mM Tris-HCI buffer (pH 8.0) containing 5 mM EDTA, 0.1 M NaCI and 1% Triton X-100
and the bacteria were disrupted by ultrasonication (out put: 4, duty cycle: 70%, 1 minute x 1 0 times). The suspension
of disrupted bacteria was centrifuged (12000 x g, 1 0 minutes) to precipitate inclusion body. Isolated inclusion body was
mixed with 50 mM Tris-HCI buffer (pH 8.0) containing 5 mM EDTA, 0.1 M NaCI and 4% Triton X-100, treated by ultra-
sonication (out put: 4, duty cycle: 50%, 30 seconds x 2 times) again and centrifuged (1 2000 x g, 1 0 minutes) to isolate
35 the desired protein as precipitate and to remove containment proteins included in the supernatant.
[0167] The inclusion body comprising the desired protein was lysed in 50 mM Tris-HCI buffer (pH 8.0) containing 6
M Urea, 5 mM EDTA and 0.1 M NaCI and applied onto Sephacryl S-300 gel filtration column (5 x 90 cm, Amersharm
Pharmacia) equilibrated with 50 mM Tris-HCI buffer (pH 8.0) containing 4M Urea, 5 mM EDTA, 0.1 M NaCI and 10 mM
mercaptoethanol at a flow rate of 5 ml/minutes to remove associated single chain Fvs with high-molecular weight. The
40 obtained fractions were analyzed with SDS-PAGE and the fractions with high purity of the protein were diluted with the
buffer used in the gel filtration up to O.D 280 =0.25. Then, the fractions were dialyzed three times against 50 mM Tris-HCI
buffer (pH 8.0) containing 5 mM EDTA, 0.1 M NaCI, 0.5 M Arg, 2 mM glutathione in the reduced form and 0.2 mM
glutathione in the oxidized form in order for the protein to be refolded. Further, the fraction was dialyzed three times
against 20 mM acetate buffer (pH 6.0) containing 0.15 M NaCI to exchange the buffer.
45 [0168] The dialysate product was applied onto Superdex 200 pg gel filtration column (2.6 x 60 cm, Amersharm
Pharmacia) equilibrated with 20 mM acetate buffer (pH 6.0) containing 0.15 M NaCI to remove a small amount of high
molecular weight protein which was intermolecularly crosslinked by S-S bonds. As shown in Fig. 24, two peaks, major
and sub peaks, were eluted after broad peaks which are expectedly attributed to an aggregate with a high molecular
weight. The analysis by SDS-PAGE (see Fig. 21 ) and the elution positions of the two peaks in the gel filtration analysis
so suggest that the major peak is of the monomer of the single chain Fv and the sub peak is of the non-covalently bound
dimer of the single chain Fv. The non-covalently bound dimer accounted for 4 percent of total single chain Fvs.
5.13 Apoptosis-inducing activity in vitro of single chain Fv derived from antibody MABL-2
55 [0169] An apoptosis-inducing action of the single chain Fv from antibody MABL-2 (MABL2-scFv) produced by the
CHO cells and E^ coli was examined according to two protocols by Annexin-V staining (Boehringer Mannheim) using
the L1210 cells (hlAP/L1210) into which human IAP gene had been introduced.
[0170] In the first protocol sample antibodies at the final concentration of 3 j^g/ml were added to 5 x 10 4 cells of
18
EP 1 327 681 A1
hlAP/L1 21 0 cell line and cultured for 24 hours. Sample antibodies, i.e., the monomer and the dimer of the single chain
Fv of MABL-2 from the CHO cells obtained in Example 5.9, the monomer and the dimer of the single chain Fvof MABL-
2 from coli obtained in Example 5.12, and the mouse IgG antibody as a control were analyzed. After culturing, the
Annexin-V staining was carried out and the fluorescence intensity thereof was measured using the FACScan apparatus
5 (BECTON DICKINSON).
[0171] In the second protocol sample antibodies at the final concentration of 3 jxg/ml were added to 5 x 1 0 4 cells of
hlAP/L121 0 cell line, cultured for 2 hours and mixed with anti-FLAG antibody (SIGMA) at the final concentration of 15
l^g/ml and further cultured for 22 hours. Sample antibodies of the monomer of the single chain Fv of MABL-2 from the
CHO cells obtained in Example 5.9 and the mouse IgG antibody as a control were analyzed. After culturing, the Annexin-
10 V staining was carried out and the fluorescence intensity thereof was measured using the FACScan apparatus.
[0172] Results of the analysis by the Annexin-V staining are shown in Figs. 25-31 . The results show that the dimers
of the single chain Fv polypeptide of MABL-2 produced in the CHO cells and E^ coli remarkably induced cell death
(Figs. 26, 27) in comparison with the control (Fig. 25), while no apoptosis-inducing action was observed in the monomers
of the single chain Fv polypeptide of MABL-2 produced in the CHO cells and E. coli (Figs. 28, 29). When anti-FLAG
15 antibody was used together, the monomer of the single chain Fv polypeptide derived from antibody MABL-2 produced
in the CHO cells induced remarkably cell death (Fig. 31 ) in comparison with the control (Fig. 30).
5.14 Antitumor effect of the monomer and the dimer of scFv/CHO polypeptide with a model mouse of human myeloma
20 (1) Quantitative measurement of human IgG in mouse serum
[0173] Measurement of human IgG (M protein) produced by human myeloma cell and contained in mouse serum
was carried out by the following ELISA. 1 00 nL of goat anti-human IgG antibody (BIOSOURCE, Lot#7902) diluted to
1 ng/mL with 0.1% bicarbonate buffer (pH 9.6) was added to each well on 96 wells plate (Nunc) and incubated at 4°C
25 overnight so that the antibody was immobilized. After blocking, 1 00 [iL of the stepwisely diluted mouse serum or human
IgG (CAPPEL, Lot#00915) as a standard was added to each well and incubated for 2 hours at a room temperature.
After washing, 100 ^L of alkaline phosphatase-labeled anti-human IgG antibody (BIOSOURCE, Lot#6202) which had
been diluted to 5000 times was added, and incubation was carried out for 1 hour at a room temperature. After washing,
asubstrate solution was added. After incubation, absorbance at 405 nm was measured using the MICROPLATE READ-
30 ER Model 3550 (BioRad). The concentration of human IgG in the mouse serum was calculated based on the calibration
curve obtained from the absorbance values of human IgG as the standard.
(2) Preparation of antibodies for administration
35 [0174] The monomer and the dimer of the scFv/CHO polypeptide were respectively diluted to 0.4 mg/mLor0.25 mg/
mL with sterile filtered PBS(-) on the day of administration to prepare samples for the administration.
(3) Preparation of a mouse model of human myeloma
40 [0175] A mouse model of human myeloma was prepared as follows. KPMM2 cells passaged in vivo (JP-Appl.
7-236475) by SCID mouse (Japan Clare) were suspended in RPMI1640 medium (GIBCO-BRL) containing 10% fetal
bovine serum (GIBCO-BRL) and adjusted to 3 x 10 7 cells/mL. 200 nL of the KPMM2 cell suspension (6 x 10 6 cells/
mouse) was transplanted to the SCID mouse (male, 6 week-old) via caudal vein thereof, which had been subcutane-
ously injected with the asialo GM1 antibody (WAKO JUNYAKU, 1 vial dissolved in 5 mL) a day before the transplantation.
45
(4) Administration of antibodies
[0176] The samples of the antibodies prepared in (2), the monomer (250 |iL) and the dimer (400 |^L), were admin-
istered to the model mice of human myeloma prepared in (3) via caudal vein thereof. The administration was started
so from three days after the transplantation of KPMM2 cells and was carried out twice a day for three days. As a control,
200 |iL of sterile filtered PBS(-) was likewise administered twice a day for three days via caudal vein. Each group
consisted of seven mice.
(5) Evaluation of antitumor effect of the monomer and the dimer of scFv/CHO polypeptide with the model mouse of
55 human myeloma
[0177] The antitumor effect of the monomer and the dimer of scFv/CHO polypeptide with the model mice of human
myeloma was evaluated in terms of the change of human IgG (M protein) concentration in the mouse serum and
19
EP 1 327 681 A1
survival time of the mice. The change of human IgG concentration was determined by measuring it in the mouse serum
collected at 24 days after the transplantation of KPMM2 cells by ELISA described in the above (1). The amount of
serum human IgG (M protein) in the serum of the PBS(-)-administered group (control) increased to about 8500 ng/mL,
whereas the amount of human IgG of the scFv/CHO dimer-administered group was remarkably low, that is, as low as
s one-tenth or less than that of the control group. Thus, the results show that the dimer of scFv/CHO strongly inhibits
the growth of the KPMM2 cells (Fig. 32). As shown in Fig. 33, a remarkable elongation of the survival time was observed
in the scFv/CHO dimer-administered group in comparison with the PBS(-)-administered group.
[0178] From the above, it is confirmed that the dimer of scFv/CHO has an antitumor effect for the human myeloma
model mice. It is considered that the antitumor effect of the dimer of scFv/CHO, the modified antibody of the invention,
10 results from the apoptosis-inducing action of the modified antibody.
5.15 Hemagglutination Test
[0179] Hemagglutination test and determination of hemagglutination were carried out in accordance with "Immuno-
15 Biochemical Investigation", Zoku-Seikagaku Jikken Koza, edited by the Biochemical Society of Japan, published by
Tokyo Kagaku Dojin.
[0180] Blood was taken from a healthy donor using heparin-treated syringes and washed with PBS(-) three times;
and then erythrocyte suspension with a final concentration of 2% in PBS(-) was prepared. Test samples were the
antibody MABL-2, the monomer and the dimer of the single chain Fv polypeptide produced by the CHO cells, and the
20 monomer and the dimer of the single chain Fv polypeptide produced by E. coli, and the control was mouse IgG (ZYMED).
For the investigation of the hemagglutination effect, round bottom 96-well plates available from Falcon were used. 50
[A. per well of the aforementioned antibody samples and 50 |iL of the 2% erythrocyte suspension were added and
mixed in the well. After incubation for 2 hours at 37°C, the reaction mixtures were stored at 4°C overnight and the
hemagglutination thereof was determined. As a control, 50 \jlL per well of PBS(-) was used and the hemagglutination
25 test was carried out in the same manner. The mouse IgG and antibody MABL-2 were employed at 0.01 , 0.1 , 1 .0, 1 0.0
or 1 00.0 ng/mL of the final concentration of the antibodies. The single chain Fvs were employed at 0.004, 0.04, 0.4,
4.0, 40.0 or 80.0 ng/mL of the final concentration and further at 160.0 |^g/mL only in the case of the dimer of the
polypeptide produced by E. coli. Results are shown in the Table 2. In the case of antibody MABL-2, the hemagglutination
was observed at a concentration of more than 0.1 ng/mL, whereas no hemagglutination was observed for both the
30 monomer and the dimer of the single chain Fv.
Table 2
Hemagglutination Test
35
mlgG
MABL-2 (intact)
Control
0.01
0.1
+
1
+++
10
+++
100
++
ng/mL
Control
0.004
0.04
0.4
4
40
80
ng/mL
40
scFv/CHO monomer
scFv/CHO-dimer
Control
0.004
0.04
0.4
4
40
80
160
ng/mL
scFv/E.coli monomer
scFv/E.coli dimer
Example 6 Modified antibody sc(Fv) 2 comprising two H chain V regions and two L chain V regions and antibody MABL-
2 scFvs having linkers with different length
6.1 Construction of plasmid expressing antibody MABL-2 sc(Fv) ?
50 —
[0181] For the preparation of a plasmid expressing the modified antibody [sc(Fv) 2 ] which comprises two H chain V
regions and two L chain V regions derived from the antibody MABL-2, the aforementioned pCHOM2, which comprises
the DNA encoding scFv derived from the MABL-2 described above, was modified by the PCR method as mentioned
below and the resulting DNA fragment was introduced into pCHOM2.
55 [0182] Primers employed for the PCR are EF1 primer (SEQ ID NO: 30) as a sense primer, which is designed to
hybridize to a DNA encoding EF1oc, and an antisense primer (SEQ ID NO: 19), which is designed to hybridize to the
DNA encoding C-terminal of the L chain V region and to contain a DNA sequence coding for a linker region, and VLLAS
20
EP 1 327 681 A1
primer containing Sail restriction enzyme recognition site (SEQ ID NO 31).
[0183] 100^1 of the PCR solution comprises 1 0 nl of 1 0 x PCR Buffer #l, 1 niM MgCI 2 , 0.2 mM dNTPs (dATP, dGTP,
dCTP and dTTP), 5 units of KOD DNA polymerase (Toyobo, Inc.), 1 fiM of each primer and 100 ng of the template
DNA (pCHOM2). The PCR solution was heated at 94°C for 30 seconds, at 50°C for 30 seconds and at 74°C for 1
s minute in order. This temperature cycle was repeated 30 times.
[0184] The PCR product was purified using the QIAquick PCR Purification Kit (QIAGEN) and digested by Sail. The
resultant DNA fragment was cloned into pBluescript KS + vector (Toyobo, Inc.). After DNA sequencing, a plasmid com-
prising the desired DNA sequence was digested by Sail and the obtained DNA fragment was connected using Rapid
DNA Ligation Kit(BOEHRINGER MANNHEIM) to pCHOM2 digested by Sail. After DNA sequencing, a plasmid com-
10 prising the desired DNA sequence is designated as "pCHOM2(Fv) 2 " (see Fig. 34). The nucleotide sequence and the
amino acid sequence of the antibody MABL-2 sc(Fv) 2 region contained in the plasmid pCHOM2(Fv) 2 are shown in
SEQ ID No. 32.
6.2 Preparation of Plasmid expressing antibody MABL-2 scFvs having linkers with various length
15
[0185] The scFvs containing linkers with different length and the V regions which are designed in the order of [H
chain]-[L chain] (hereinafter "HL") or [L chain]-[H chain] (hereinafter "LH") were prepared using, as a template, cDNAs
encoding the H chain and the L chain derived from the MABL-2 as mentioned below.
[0186] To construct HL type scFv the PCR procedure was carried out using pCHOM2(Fv) 2 as a template. In the PCR
20 step, a pair of CFHL-F1 primer (SEW ID NO: 33) and CFHL-R2 primer (SEQ ID NO: 34) or a pair of CFHL-F2 primer
(SEQ ID NO: 35) and CFHL-R1 primer (SEQ ID NO: 36) and KOD polymerase were employed. The PCR procedure
was carried out by repeating 30 times the temperature cycle consisting of 94°C for 30 seconds, 60°C for 30 seconds
and 72°C for 1 minute in order to produce a cDNA for the H chain containing a leader sequence at 5'-end or a cDNA
for the L chain containing FLAG sequence at 3'-end thereof. The resultant cDNAs for the H chain and the L chain were
25 mixed and PCR was carried out by repeating 5 times the temperature cycle consisting of 94°C for 30 seconds, 60°C
for 30 seconds and 72°C for 1 minute in order using the mixture as templates and the KOD polymerase. To the reaction
mixture were added CFHL-F1 and CFHL-R1 primers and then the PCR reaction was performed by repeating 30 times
of the aforementioned temperature cycle to produce acDNA for HL-0 type without a linker.
[0187] To construct LH type scFv, the PCR reaction was carried out using, as a template, pGEM-M2L and pGEM-M2H
30 which contain cDNAs encoding the L chain V region and the H chain V region from the antibody MABL-2, respectively
(see JP- Appl. 11-63557). A pair of T7 primer (SEQ ID NO: 37) and CFLH-R2 primer(SEQ ID NO: 38) or a pair of
CFLH-F2 primer (SEQ ID NO: 39) and CFLH-R1 (SEQ ID NO: 40) and the KOD polymerase (Toyobo Inc.) were em-
ployed. The PCR reaction was performed by repeating 30 times the temperature cycle consisting of 94°C for 30 sec-
onds, 60°C for 30 seconds and 72°C for 1 minute in sequential order to produce a cDNA of an L chain containing a
35 leader sequence at 5'-end or a cDNA of an H chain containing FLAG sequence at 3'-end thereof. The resultant cDNAs
of the L chain and the H chain were mixed and PCR was carried out using this mixture as templates and the KOD
polymerase by repeating 5 times the temperature cycle consisting of 94°C for 30 seconds, 60°C for 30 seconds and
72°C for 1 minute in order. To the reaction mixture were added T7 and CFLH-R1 primers and the reaction was performed
by repeating 30 times of the aforementioned temperature cycle. The reaction product was used as a template and
40 PCR was carried out using a pair of CFLH-F4 primer (SEQ ID NO: 41 ) and CFLH-R1 primer by repeating 30 times the
temperature cycle consisting of 94°C for 30 seconds, 60°C for 30 seconds and 72°C for 1 minute in order to produce
a cDNA of LH-0 type without a linker.
[0188] The resultant cDNAs of LH-0 and HL-0 types were digested by EcoRI and BamHI restriction enzymes (Takara
Shuzo) and the digested cDN As were introduced into an expression plasmid INPEP4for mammalian cells using Ligation
45 High (Toyobo Inc.), respectively. Competent E. coli JM1 09 (Nippon Gene) was transformed with each plasmid and the
desired plasmids were isolated from the transformed E^ coli using QIAGEN Plasmid Maxi Kit (QUIAGEN). Thus plas-
mids pCF2LH-0 and pCF2HL-0 were prepared.
[0189] To construct the expression plasmids of HL type containing linkers with different size, pCF2HL-0, as a tem-
plate, and CFHL-X3 (SEQ ID NO: 42), CFHL-X4 (SEQ ID NO: 43), CFHL-X5 (SEQ ID NO: 44), CFHL-X6 (SEQ ID NO:
so 45) or CFHL-X7 (SEQ ID NO: 46), as a sense primer, and BGH-1 (SEQ ID NO: 47) primer, as an antisense primer,
which is complementary with the vector sequence were employed. PCR reaction was carried out using the KOD
polymerase by repeating 30 times the temperature cycle consisting of 94°C for 30 seconds, 60°C for 30 seconds and
72°C for 1 minute in order and the reaction products were digested by restriction enzymes Xhol and BamHI (Takara
Shuzo). The digested fragments were introduced between Xhol and BamHI sites in the pCF2HL-0 using Ligation High
55 (Toyobo Inc.), respectively. Competent E^ coli JM1 09 was transformed with each plasmid and the desired plasmids
were isolated from the transformed E^coli by using Qiagen Plasmid Maxi kit. Thus expression plasmids pCF2HL-3,
pCF2HL-4, pCF2HL-5, pCF2HL-6 and pCF2HL-7 were prepared.
[0190] To construct expression plasmid for the transient expression in COS7 cells the plasmids pCF2HL-0, pCF2HL-
21
EP 1 327 681 A1
3, pCF2HL-4, pCF2HL-5, pCF2HL-6 and pCF2HL-7 were digested by restriction enzymes EcoRI and BamHI (Takara
Shuzo) and the resultant fragments of approximately 800 bp were purified with agarose gel electrophoresis. The ob-
tained fragments were introduced between EcoRI and BamHI sites in an expression plasmid pCOS1 for the expression
in mammalian cells by using Ligation High (Toyobo Inc.), respectively. Competent E_ coli DH5oe (Toyobo Inc.) was
s transformed with each plasmid and the desired plasmids were isolated from the transformed coli using Qiagen
Plasmid Maxi kit. Thus the expression plasmids CF2HL-0/pCOS1 , CF2HL-3/pCOS1 , CF2HL-4/pCOS1 , CF2HL-
5/pCOS1, CF2HL-6/pCOS1 and CF2HL-7/pCOS1 were prepared.
[0191] As a typical example of these plasmids, the construction of the plasmid CF2HL-0/pCOS1 is illustrated in Fig.
35 and the nucleotide sequence and the amino acid sequence of MABL2-scFv <HL-0> contained in the plasmid are
10 shown in SEQ ID No. 48. Nucleotide sequences and amino acid sequences of the linker regions in these plasmids are
also shown in Fig. 36.
[0192] To construct the expression plasmids of LH type containing linkers with different size, pCF2LH-0, as a tem-
plate, and CFLH-X3 (SEQ ID NO: 49), CFLH-X4 (SEQ ID NO: 50), CFLH-X5 (SEQ ID NO: 51), CFLH-X6 (SEQ ID NO:
52) or CFLH-X7 (SEQ ID NO: 53), as a sense primer, and BGH-1 primer, as an antisense primer, which is compte-
rs mentary with the vector sequence were employed. PCR reaction was carried out using the KOD polymerase by re-
peating 30 times the temperature cycle consisting of 94°C for 30 seconds, 60°C for 30 seconds and 72°C for 1 minute
in order and the reaction products were digested by restriction enzymes Xhol and BamHI. The digested fragments
were introduced into the pCF2LH-0 between Xhol and BamHI sites using Ligation High, respectively. Competent E^
coli DH5oc (Toyobo Inc.) was transformed with each plasmid and the desired plasmids were isolated from the trans-
20 formed E. coli using Qiagen Plasmid Maxi kit. Thus expression plasmids pCF2LH-3, pCF2LH-4, pCF2LH-5, pCF2LH-
6 and pCF2LH-7 were prepared.
[0193] To construct expression plasmid for the transient expression in COS7 cells the plasmids pCF2LH-0, pCF2LH-
3, pCF2LH-4, pCF2LH-5, pCF2LH-6 and pCF2LH-7 were digested by restriction enzymes EcoRI and BamHI (Takara
Shuzo) and the resultant fragments of approximately 800 bp were purified with agarose gel electrophoresis. The ob-
25 tained fragments were introduced between Xhol and BamHI sites in an expression plasmid pCOS1 for the expression
in mammalian cells by using the Ligation High, respectively. Competent E. coli DH5oc (Toyobo Inc.) was transformed
with each plasmid and the desired plasmids were isolated from the transformed EL coli using the Qiagen Plasmid Maxi
kit. Consequently, the expression plasmids CF2LH-0/pCOS1 , CF2LH-3/pCOS1 , CF2LH-4/pCOS1 , CF2LH-5/pCOS1 ,
CF2LH-6/pCOS1 and CF2LH-7/pCOS1 were prepared.
30 [0194] As a typical example of these plasmids, the construction of the plasmid CF2LH-0/pCOS1 is illustrated in Fig.
37 and the nucleotide sequence and the amino acid sequence of MABL2-scFv <LH-0> contained in the plasmid are
shown in SEQ ID No. 54. Nucleotide sequences and amino acid sequences of the linker regions in these plasmids are
also shown in Fig. 38.
35 6.3 Expression of scFvs and sc(Fv) ? in COS7 cells
(1) Preparation of culture supernatant using serum-containing culture medium
[0195] The HL type and LH type of scFvs and sc(Fv) 2 were transiently expressed in COS7 cells (JCRB9127, Japan
40 Health Sciences Foundation). COS7 cells were subcultured in DMEM media (GIBCO BRL) containing 1 0% fetal bovine
serum (HyClone) at 37°C in carbon dioxide atmosphere incubator. The COS7 cells were transfected with CF2HL-0, 3
~ 7/pCOS1, or CF2LH-0, 3 ~ 7/pCOS1 prepared in Example 6.2 or pCHOM2(Fv) 2 vectors by electroporation using
the Gene Pulser apparatus (BioRad). The DNA (10 ng) and 0.25 ml of 2 x 10 7 cells/ml in DMEM culture medium
containing 10% FBS and 5 mM BES (SIGMA) were added to a cuvette. After standing for 10 minutes the mixtures
45 were treated with pulse at 0.1 7kV, 950|iF of electric capacity. After the restoration for 1 0 minutes at room temperature,
the electroporated cells were transferred into the DMEM culture medium (1 0%FBS) in 75 cm 3 flask. After culturing for
72 hours, the culture supernatant was collected and centrifuged to remove cell fragments. The culture supernatant
was subjected to the filtration using 0.22 |^m bottle top filter (FALCON) to obtain the culture supernatant (hereinafter
"CM").
50
(2) Preparation of culture supernatant using serum-free culture medium
[0196] Cells transfected in the same manner as (1) were transferred to the DMEM medium (10% FBS) in 75 cm 3
flask and cultured overnight. After the culture, the supernatant was discarded and the cells were washed with PBS and
55 then added to CHO-S-SFM II medium (GIBCO BRL). After culturing for 72 hours, the culture supernatant was collected,
centrifuged to remove cell fragments and filtered using 0.22 urn bottle top filter (FALCON) to obtain CM.
22
EP 1 327 681 A1
6.4 Detection of scFvs and sc(Fv) P in CM of COS7
[0197] The various MABL2-scFVs and sc(Fv) 2 in CM of COS7 prepared in the aforementioned Example 6.3 (2) were
detected by Western Blotting method.
s [0198] Each CM of COS7 was subjected to SDS-PAGE electrophoresis and transferred to REINFORCED NC mem-
brane (Schleicher & Schuell). The membrane was blocked with 5% skim milk (Morinaga Nyu-gyo) and washed with
TBS. Then an anti-FLAG antibody (SIGMA) was added thereto. The membrane was incubated at room temperature
and washed. A peroxidase labeled mouse IgG antibody (Jackson Immuno Research) was added. After incubating and
washing at room temperature, the substrate solution (Kirkegaard Perry Laboratories) was added to develop color (Fig.
10 39).
6.5 Flow cytometry
[0199] Flow cytometry was performed using the culture supernatants of COS7 cells prepared in Example 6.3 (1 ) to
15 measure the binding of the MABL2-scFVs and sc(Fv) 2 to human Integrin Associated Protein (IAP) antigen. The culture
supernatants to be tested or a culture supernatant of COS7 cells as a control was added to 2 x 1 05 cells of the mouse
leukemia cell line L1 21 0 expressing human IAP. After incubating on ice and washing, 10ng/mL of the mouse anti-FLAG
antibody (SIGMA) was added and then the cells were incubated and washed. Then, the FITC labeled anti-mouse IgG
antibody (BECTON DICKINSON) was added thereto and the cells were incubated and washed again. The fluorescence
20 intensity was measured using the FACScan apparatus (BECTON DICKINSON). The results of the flow cytometry show
that the MABL2-scFvs having linkers with different length and the sc(Fv) 2 in the culture supernatants of COS7 have
high affinity to human IAP (see Figs. 40a and 40b).
6.6 Apoptosis-inducing Effect in vitro
25
[0200] An apoptosis-inducing action of the culture supernatants of COS7 prepared in Example 6.3 (1 ) was examined
by Annexin-V staining (Boehringer Mannheim) using the L1210 cells transfected with human IAP gene (hlAP/L1 21 0).
[0201] To 5 x 10 4 cells of the hlAP/L1210 cells were added the culture supernatants of COS7 cells transfected with
each vectors or a culture supernatant of COS7 cells as a control at 1 0% of the final concentration and the mixtures
30 were cultured for 24 hours. Then, the Annexin-V/PI staining was performed and the fluorescence intensity was meas-
ured using the FACScan apparatus (BECTON DICKINSON). The results revealed that scFvs <HL3, 4, 6, 7, LH3, 4, 6,
7> and sc(Fv) 2 in CM of COS7 induced remarkable cell death of hlAP/L121 0 cells. These results are shown in Fig. 41 .
6.7 Construction of vectors for the expression of scFvs and sc(Fv) ? in CHO cells
35
[0202] To isolate and purify MABL2-scFvs and sc(Fv) 2 from culture supernatant, the expression vectors for express-
ing in CHO cells were constructed as below.
[0203] The EcoRI-BamHI fragments of pCF2HL-0, 3 ~ 7, and pCF2LH-0, 3 ~ 7 prepared in Example 6.2 were
introduced between EcoRI and BamHI sites in an expression vector pCHOI for CHO cells using the Ligation High.
40 Competent E^ coli DH5a was transformed with them. The plasmids were isolated from the transformed E^ coli using
QIAGEN Plasmid Midi kit (QIAGEN) to prepare expression plasmids pCHOM2HL-0, 3 ~ 7, and pCHOM2LH-0, 3 ~ 7.
6.8 Production of CHO cells expressing MABL2-scFvs <HL-0, 3 ~ 7>, MABL2-scFvs <LH-0, 3 ~ 7> and sc(Fv) 3 and
preparation of the culture supernatants thereof
45
[0204] CHO cells were transformed with each of the expression plasmids pCHOM2HL-0, 3 ~ 7, and pCHOM2LH-
0, 3 ~ 7, constructed in Example 6.7 and pCHOM2(Fv) 2 vector to prepare the CHO cells constantly expressing each
modified antibody. As atypical example thereof, the production of the CHO cells constantly expressing MABL2-scFv
<HL-5> or sc(Fv) 2 is illustrated as follows.
so [0205] The expression plasmids pCHOM2HL-5 and pCHOM2(Fv) 2 were linearized by digesting with a restriction
enzyme Pvul and subjected to transfection to CHO cells by electroporation using Gene Pulser apparatus (BioRad).
The DNA (1 0 ng) and 0.75 ml of PBS with 1 x 1 0 7 cells/ml were added to a cuvette and treated with pulse at 1 .5 kV,
25 \\f of electric capacity. After the restoration for 10 minutes at room temperature, the electroporated cells were
transferred into nucleic acid-containing oc-MEM culture medium (GIBCO BRL) containing 10% fetal bovine serum and
55 cultured. After culturing overnight, the supernatant was discarded. The cells were washed with PBS and added to
nucleic acid-free oe-MEM culture medium (GIBCO BRL) containing 10% fetal bovine serum. After culturing for two
weeks, the cells were cultured in a medium containing 1 0 nM (final concentration) methotrexate (SIGMA), then 50 nM
and 1 00 nM methotrexate. The resultant cells were cultured in serum-free CHO-S-SFM II medium (GIBCO BRL) in a
23
EP 1 327 681 A1
roller bottle. The culture supernatant was collected, centrifuged to remove cell fragments and filtered using a filter with
0.22 urn of pore size to obtain CM, respectively.
[0206] According to the above, CHO cells which constantly express MABL2-scFvs <HL-0, -3, -4, -6, -7> and <LH-
0, -3,-4, -5, -6, -7> and CMs thereof were obtained.
5
6.9 Purification of dimer of MABL2-scFv <HL-5> and sc(Fv) ?
[0207] The MABL2-scFv <HL-5> and the sc(Fv) 2 were purified from CMs prepared in Example 6.8 by two types of
purification method as below.
10
Purification Method 1>
[0208] HL-5 and sc(Fv) 2 were purified by the anti-FLAG antibody affinity column chromatography utilizing the FLAG
sequence located at C-terminal of the polypeptides and by gel filtration. One liter of CM as obtained in 6.8 was applied
15 onto a column (7.9ml) prepared with anti-FLAG M2 Affinity gel (SIGMA) equilibrated with 50 mM Tris-HCI buffer (TBS,
pH 7.5) containing 1 50 mM NaCI. After washing the column with TBS, the scFv was eluted by 0.1 M glycine-HCI buffer,
pH 3.5. The resultant fractions were analyzed by SDS-PAGE and the elution of the scFv was confirmed. The scFv
fraction was mixed with Tween 20 up to 0.01% of the final concentration and concentrated using Centricon-10 (MILI-
PORE). The concentrate was applied onto TSKgel G3000SWG column (7.5 x 600 mm) equilibrated with 20 mM acetate
20 buffer (pH 6.0) containing 1 50 mM NaCI and 0.01 % Tween 20. At 0.4 mL/minute of the flow rate, the scFv was detected
by the absorption at 280 nm. The HL-5 was eluted as the major fraction in the position of the dimer and the sc(Fv) 2
was eluted in the position of the monomer.
Purification Method 2>
25
[0209] HL-5 and sc(Fv) 2 were purified using three steps comprising ion exchange chromatography, hydroxyapatite
and gel filtration. In the ion exchange chromatography, Q sepharose fast flow column (Pharmacia) was employed for
HL-5 and SP-sepharose fast flow column was employed for sc(Fv) 2 . In and after the second step, HL-5 and sc(Fv) 2
were processed by the same procedure.
30
First step for HL-5
[0210] CM of HL-5 was diluted to two times with 20 mM Tris-HCI buffer (pH 9.0) containing 0.02% Tween 20 and
then the pH was adjusted to 9.0 with 1 M Tris. The solution was applied onto Q Sepharose fast flow column equilibrated
35 with 20 mM Tris-HCI buffer (pH 8.5) containing 0.02% Tween 20. A polypeptide adsorbed to the column was eluted by
a linear gradient of NaCI in the same buffer, from 0.1 to 0.55 M. Monitoring the eluted fractions by SDS-PAGE, the
fractions containing HL-5 were collected and subjected to hydroxyapatite of the second step.
First step for sc(Fv) ?
40
[0211] CM of the sc(Fv) 2 was diluted to two times with 20mM acetate buffer (pH 5.5) containing 0.02% Tween 20
and its pH was adjusted to 5.5 with 1 M acetic acid. The solution was applied onto a SP-Sepharose fast flow column
equilibrated with 20 mM acetate buffer (pH 5.5) containing 0.02% Tween 20. A polypeptide adsorbed to the column
was eluted by a linear gradient of NaCI in the buffer, from 0 to 0.5 M. Monitoring the eluted fractions by SDS-PAGE,
45 the fractions containing the sc(Fv) 2 were collected and subjected to hydroxyapatite of the second step.
Second step: Hydroxyapatite chromatography of HL-5 and sc(Fv) 2
[0212] The fractions of HL-5 and sc(Fv) 2 obtained in the first step were separately applied onto the hydroxyapatite
so column (Type I, BIORAD) equilibrated with 10 mM phosphate buffer containing 0.02% Tween 20, pH 7.0. After washing
the column with the same buffer, polypeptides adsorbed to the column were eluted by a linear gradient of the phosphate
buffer up to 0.5 M. Monitoring the eluted fractions by SDS-EAGE, the fractions containing the desired polypeptides
were collected.
55 Third step: Gel filtration of HL-5 and sc(Fv) 2
[0213] Each fraction obtained at the second step was separately concentrated with CentriPrep-1 0 (MILIPORE) and
applied onto a Superdex 200 column (2.6 x 60 cm, Pharmacia) equilibrated with 20 mM acetate buffer (pH 6.0) con-
EP 1 327 681 A1
taining 0.02% Tween 20 and 0.15 M NaCI. HL-5 was eluted in the position of the dimer, and sc(Fv)HL-5 and sc(Fv) 2
were eluted in the position of the monomer as a major peek respectively.
[0214] Since the monomer of HL-5 was hardly detected by both purification methods, it is proved that the dimers of
single chain Fvs are formed in high yields when the linker for the single chain Fv contains around 5 amino acids.
s Furthermore, the dimer of HL-5 and the sc(Fv) 2 were stably preserved for a month at 4°C after the purification.
6.1 0 Evaluation of the binding activity of purified dimer of scFv <HL-5> and sc(Fv)? against antigen
[0215] Flow cytometry was performed using the purified dimer of MABL2-scFv <HL-5> and the purified sc(Fv) 2 in
10 order to evaluate the binding to human Integrin Associated Protein (IAP) antigen. 10|ig/ml of the purified dimer of
MABL2-scFv <HL-5>, the purified sc(Fv) 2 , the antibody MABL-2 as a positive control or a mouse IgG (Zymed) as a
negative control was added to 2 x 10 5 cells of the mouse leukemiacell line L121 0 expressing human IAP (hlAP/L1210)
or the cell line L121 0 transformed with pCOS1 (pCOS1/L121 0) as a control. After incubating on ice and washing, 1 0\\qI
mL of the mouse anti-FLAG antibody (SIGMA) was added and then the cells were incubated and washed. FITC labeled
15 anti-mouse IgG antibody (BECTON DICKINSON) was added thereto and the cells were incubated and washed again.
Then the fluorescence intensity was measured using the FACScan apparatus (BECTON DICKINSON).
[0216] Since the purified dimer of MABL2-scFv <HL-5> and the purified sc(Fv) 2 were specifically bound to hlAP/
L1 210 cells, it is confirmed that the dimer of scFv <HL-5> and the sc(Fv) 2 have high affinity to human IAP (see Fig. 42).
20 6.11 Apoptosis-inducing activity in vitro of purified dimer of scFv <HL-5> and sc(Fv) ?
[0217] An apoptosis-inducing action of the purified dimer of MABL2-scFv <HL-5> and the purified sc(Fv) 2 were ex-
amined by Annexin-V staining (Boehringer Mannheim) using the L1210 cells (hlAP/L1210) in which human IAP gene
had been introduced and cells of human leukemic cell line CCRF-CEM.
25 [0218] Different concentrations of the purified dimer of MABL2-scFv <HL-5>, the purified MABL2-sc(Fv) 2 , the anti-
body MABL-2 as a positive control or a mouse IgG as a negative control were added to 5 x 10 4 cells of hlAP/L1210
cell line or 1 x 10 5 cells of CCRF-CEM cell line. After culturing for 24 hours, the Annexin-V staining was carried out
and the fluorescence intensity thereof was measured using the EACScan apparatus (BECTON DICKINSON). As a
result the dimer of MABL2-scFv <HL-5> and the MABL2-sc(Fv) 2 remarkably induced cell death of hHIAP/L1210 and
30 CCRF-CEM in concentration-dependent manner (see Fig. 43). As a result it was shown that the dimer of MABL2-scFv
<HL-5> and MABL2-sc(Fv) 2 , had improved efficacy of inducing apoptosis compared with original antibody MABL-2.
6.12 Hemagglutination Test of the purified dimer of scFv <HL-5> and the sc(Fv) ?
35 [0219] Hemagglutination test was carried out using different concentrations of the purified dimer of scFv <HL-5> and
the purified sc(Fv) 2 in accordance with Example 5.15.
[0220] The hemagglutination was observed with the antibody MABL-2 as a positive control, whereas no hemagglu-
tination was observed with both the single chain antibody MABL2-SC(Fv) 2 and the MABL2-scFv <HL-5>. Further, there
was no substantial difference in the hemagglutination between two buffers employed with the antibody MABL-2. These
40 results are shown in Table 3.
45
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EP 1 327 681 A1
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26
EP 1 327 681 A1
6.13 Antitumor effect of the purified dimer of scFv <HL-5> and the sc(Fv) P for a model mouse of human myeloma
[0221] The antitumor effects were tested for the dimer of scFv <HL-5> and the sc(Fv) 2 prepared and purified in
Examples 6.8 and 6.9. The test was performed by using the mouse model for human myeloma produced in Example
s 5.1 and determining the amount of M protein produced by human myeloma cells in the mouse serum using ELISA and
examining survival time of the mice. Then, the antitumor effects of the dimer of scFv <HL-5> and the sc(Fv) 2 were
evaluated in terms of the change of the amount of M protein in the mouse serum and the survival time of the mice.
[0222] In the test, the HL-5 and the sc(Fv) 2 were employed as a solution at 0.01 , 0.1 or 1 mg/mL in vehicle consisting
of 150 mM NaCI, 0.02% Tween and 20 mM acetate buffer, pH 6.0 and administered to the mice at 0.1 , 1 or 1 0 mg/kg
10 of dosage. Control group of mice were administered only with the vehicle.
[0223] The mouse serum was gathered 26 days after the transplantation of the human myeloma cells and the amount
of M protein in the serum was measured using ELISA according to Example 5.14. As a result, the amount of M protein
in the serum of both mice groups administered with HL-5, the dimer and the sc(Fv) 2 decreased in dose-dependent
manner (see Fig. 44). Furthermore, a significant elongation of the survival time was observed in both groups admin-
15 istered with the HL-5 (Fig. 45) and with the sc(Fv) 2 (Fig. 46) in comparison with the control group administered with
the vehicle. These results show that the HL-5 and the sc(Fv) 2 of the invention have excellent antitumor effect in vivo .
Example 7
20 Single chain Fv comprising H chain V region and L chain V region of human antibody 12B5 against human MPL
[0224] A DNA encoding V regions of human monoclonal antibody 12B5 against human MPL was constructed as
follows:
25 7.1 Construction of a gene encoding H chain V region of 12B5
[0225] The gene encoding H chain V region of human antibody 12B5 binding to human MPL was designed by con-
necting the nucleotide sequence of the gene thereof (SEQ ID NO: 55) at the 5'-end to the leader sequence (SEQ ID
NO: 56) originated from human antibody gene (Eur. J. Immunol. 1 996; 26: 63-69). The designed nucleotide sequence
30 was divided into four oligonucleotides having overlapping sequences of 1 5 bp each (1 2B5VH-1 , 1 2B5VH-2, 1 2B5VH-
3, 12B5VH-4). 12B5VH-1 (SEQ ID NO: 57) and 12B5VH-3 (SEQ ID NO: 59) were synthesized in the sense direction,
and 12B5VH-2 (SEQ ID NO: 58) and 12B5VH-4 (SEQ ID NO: 60) in the antisense direction, respectively. After assem-
bling each synthesized oligonucleotide by respective complementarity, the outside primers (1 2B5VH-S and 1 2B5VH-A)
were added to amplify the full length of the gene. 12B5VH-S (SEQ ID NO: 61) was designed to hybridize to 5'-end of
35 the leader sequence by the forward primer and to have Hind III restriction enzyme recognition site and Kozak sequence,
and 12B5VH-A (SEQ ID NO: 62) was designed to hybridize to the nucleotide sequence encoding C-terminal of H chain
V region by the reverse primer and to have a splice donor sequence and BamHI restriction enzyme recognition site,
respectively.
[0226] 1 0Ojul of the PCR solution containing 1 0jil of 1 0 x PCR Gold Buffer II, 1 .5mM MgCI 2 , 0.08mM dNTPs (dATP,
40 dGTP, dCTP, dTTP), 5 units of DNA-polymerase AmpliTaq Gold (all by PERKIN ELMER) and each 2.5 p mole of each
synthesized oligonucleotide (12B5VH-1 to -4) was heated at 94°C of the initial temperature for 9 minutes, at 94°C for
2 minutes, at 55°C for 2 minutes and 72°C for 2 minutes. After repeating the cycle two times each 1 00 pmole of external
primer 12B5VH-S and 12B5VH-A was added. The mixture was subjected to the cycle consisting of at 94°C for 30
seconds, at 55°C for 30 seconds and 72°C for 1 minute 35 times and heated at 72°C for further 5 minutes.
45 [0227] The PCR product was purified by 1 .5% low-melting-temperature agarose gel (Sigma), digested by restriction
enzymes BamHI and Hind III, and cloned into expression vector HEF-gy1 for human H chain. After determining the
DNA sequence the plasmid containing the correct DNA sequence was named HEF-12B5H-gy1 .
[0228] The HEF-12B5H-gy1 was digested by restriction enzymes EcoRI and BamHI to produce the gene encoding
12B5VH which was then cloned into an expression vector pCOS-Fd for human Fab H chain to produce pFd-12B5H.
so The expression vector for human Fab H chain was constructed by amplifying the DNA (SEQ ID NO: 63) containing the
intron region existing between the genes encoding human antibody H chain V region and the constant region, and the
gene encoding a part of the constant region of human H chain by PCR, and inserting the PCR product into animal cell
expression vector pCOS1 . The human H chain constant region was amplified for the gene under the same conditions
mentioned above using as the template HEF-gy1 , as the forward primer G1 CH1 -S (SEQ ID NO: 64) which was designed
55 to hybridize to 5'-end sequence of intron 1 and to have restriction enzyme recognition sites EcoRI and BamHI and as
the reverse primer G1CH1-A (SEQ ID NO: 65) which was designed to hybridize to 3'-end DNA of human H chain
constant region CH1 domain and to have a sequence encoding a part of hinge region, two stop codons and restriction
enzyme recognition site Bg1 II.
27
EP 1 327 681 A1
[0229] The nucleotide sequence and amino acid sequence of the reconstructed 1 2B5H chain variable region which
were included in plasmids HEF-12B5H-gy1 and pFd-12B5H are shown in SEQ ID NO: 66.
7.2 Construction of the gene encoding 12B5 L chain V region
5
[0230] The gene encoding L chain V region of human antibody 1 2B5 binding to human MPL was designed by con-
necting the nucleotide sequence of gene (SEQ ID NO: 67) at the 5'-end to the leader sequence (SEQ ID NO: 68)
originated from human antibody gene 3D6 (Nuc. Acid Res. 1 990: 1 8; 4927). In the same way as mentioned above the
designed nucleotide sequence was divided into four oligonucleotides having overlapping sequences of 15 bp each
10 (12B5VL-1 , 12B5VL-2, 12B5VL-3, 12B5VL-4) and synthesized respectively. 12B5VL-1 (SEQ ID NO: 69) and 12B5VL-
3 (SEQ ID NO: 71) had sense sequences, end 12B5VL-2 (SEQ ID NO: 70) and 12B5VL-4 (SEQ ID NO: 72) had
antisense sequences, respectively. Each of the synthesized oligonucleotides was assembled by respective comple-
mentarity and mixed with the external primer (1 2B5VL-S and 12B5VL-A) to amplify the full length of the gene. 12B5VL-S
(SEQ ID NO: 73) was designed to hybridize to 5'-end of the leader sequence by the forward primer and to have Hind
15 Ml restriction enzyme recognition site and Kozak sequence. 12B5VL-A (SEQ ID NO: 74) was designed to hybridize to
the nucleotide sequence encoding C-terminal of L chain V region by the reverse primer and to have a splice donor
sequence and BamHI restriction enzyme recognition site.
[0231] Performing the PCR as mentioned above, the PCR product was purified by 1.5% low-melting-temperature
agarose gel (Sigma), digested by restriction enzymes BamHI and Hind III, and cloned into an expression vector HEF-
20 gK for human L chain. After determining the DNA sequence the plasmid containing the correct DNA sequence was
named HEF-12B5L-gK. The nucleotide sequence and amino acid sequence of the reconstructed 12B5 L chain V region
which were included in plasmid HEF-12B5L-gK are shown in SEQ ID NO:75.
7.3 Production of reconstructed 12B5 single chain Fv (scFv)
25
[0232] The reconstructed 12B5 antibody single chain Fv was designed to be in the order of 12B5VH-linker-12B5VL
and to have FLAG sequence (SEQ ID NO: 76) at C-terminal to facilitate the detection and purification. The reconstructed
12B5 single chain Fv (sc1 2B5) was constructed using a linker sequence consisting of 15 amino acids represented by
(Gly 4 Ser) 3 .
30
(1 ) Production of the reconstructed 1 2B5 single chain Fv using the linker sequence consisting of 1 5 amino acids
[0233] The gene encoding the reconstructed 12B5 antibody single chain Fv, which contained the linker sequence
consisting of 15 amino acids, was constructed by connecting 12B5 H chain V region, linker region and 12B5 L chain
35 v region which was amplified by PCR respectively. This method is schematically shown in Fig. 47. Six PCR primers
(A-F) were used for production of the reconstructed 1 2B5 single chain Fv. Primers A, C, and E had sense sequences,
and primers B, D, and F had antisense sequences.
[0234] The forward primer 12B5-S (Primer A, SEQ ID NO: 77) for H chain V region was designed to hybridize to 5'-
end of H chain leader sequence and to have EcoRI restriction enzyme recognition site. The reverse primer HuVHJ3
40 (Primer B, SEQ ID NO: 78) for H chain V region was designed to hybridize to DNA encoding C-terminal of H chain V
region.
[0235] The forward primer RHuJH3 (Primer C, SEQ ID NO: 79) for the linker was designed to hybridize to DNA
encoding the N-terminal of the linker and to overlap DNA encoding the C-terminal of H chain V region. The reverse
primer RHuVKI (Primer D, SEQ ID NO: 80) for the linker was designed to hybridize to DNA encoding the C-terminal
45 of the linker and overlap DNA encoding the N-terminal of L chain V region.
[0236] The forward primer HuVK1.2 (Primer E, SEQ ID NO: 81) for L chain V region was designed to hybridize to
DNA encoding the N-terminal of L chain V region. The reverse primer 12B5F-A for L chain V region (Primer F, SEQ
ID NO: 82) was designed to hybridize to DNA encoding C-terminal of L chain V region and to have the sequence
encoding FLAG peptide (Hopp, T. P. et al., Bio/Technology, 6, 1204-1210, 1988), two transcription stop codons and
so Notl restriction enzyme recognition site.
[0237] In the first PCR step, three reactions A-B, C-D, and E-F were performed, and the three PCR products obtained
from the first step PCR were assembled by respective complementarity. After adding primers A and F the full length
DNA encoding the reconstructed 12B5 single chain Fv having the linker consisting of 15 amino acids was amplified
(the second PCR). In the first step PCR, the plasmid HEF-12B5H-gy1 (see Example 7. 1 ) encoding the reconstructed
55 12B5 H chain V region, pSCFVT7-hM21 (humanized ONS-M21 antibody) (Ohtomo et al., Anticancer Res. 18 (1998),
4311-4316) containing DNA (SEQ ID NO: 83) encoding the linker region consisting of Gly Gly Gly Gly Ser Gly Gly Gly
Gly Ser Gly Gly Gly Gly Ser (Huston et al., Proc. Natl. Acad. Sci. USA, 85, 5879-5883, 1988) and the plasmid HEF-
12B5L-gtc (see Example 7. 2) encoding the reconstructed 12B5 L chain V region were used as templates, respectively.
28
EP 1 327 681 A1
[0238] 50|il of PCR solution for the first step contained 5|il of 10 x PCR Gold Buffer II, 1.5mM MgCI 2 , 0.08mM dNTPs,
5 units of DNA polymerase AmpliTaq Gold (all by PERKIN ELMER), each 1 00 pmole of each primer and 100ng of each
template DNA. The PCR solution was heated at 94°C of the initial temperature for 9 minutes, at 94 for 30 seconds,
55°C for 30 seconds and 72°C for 1 minute. After repeating the cycle 35 times the reaction mixture was further heated
s 72°C for 5 minutes.
[0239] The PCR products A-B, C-D, and E-F were assembled by the second PCR. PCR mixture solution for the
second step of 98^1 containing as the template 1^1 of the first PCR product A-B, 0.5^1 of PCR product C-D and 1 jxl of
PCR product E-F, 10^1 of 10x PCR Gold Buffer II, 1.5mM MgCI 2 , 0.08mM dNTPs, 5 units of DNA polymerase AmpliTaq
Gold (all by PERKIN ELMER) was heated at 94°C of the initial temperature for 9 minutes, at 94°C for 2 minutes, at
10 65°C for 2 minutes and 72°C for 2 minutes. After repeating the cycle two times, each 1 00 pmole of each of primers A
and F were added. After repeating the cycle consisting of at 94°C for 30 seconds, 55°C for 30 seconds and 72°C for
1 minute 35 times, the reaction mixture was heated at 72°C for 5 minutes.
[0240] The DNA fragments produced by the second PCR were purified using 1 .5% low-melting-temperature agarose
gel, digested by EcoRI and Notl, and cloned into pCHOI vector and pCOS1 vector (Japanese Patent Application No.
15 8-255196). The expression vector pCHOI was a vector constructed by deleting the antibody gene from DHFR-AE-
rvH-PM1-f (see W092/19759) by EcoRI and Smal digestion, and connecting to EcoRI-Notl-BamHI Adaptor (TAKARA
SHUZO). After determining the DNA sequence the plasmids containing the DNA fragment encoding the correct amino
acid sequence of reconstructed 12B5 single chain Fv were named pCHO-sc12B5 and pCOS-sc12B5. The nucleotide
sequence and amino acid sequence of the reconstructed 1 2B5 single chain Fv included in the plasmids pCHO-sc1 2B5
20 and pCOS-sc12B5 are shown in SEQ ID NO: 84.
7.4 Expression of antibody 12B5 (IgG, Fab) and single chain Fv polypeptide by animal cell
[0241] Antibody 12B5 (IgG, Fab) and single chain Fv derived from antibody 12B5 were expressed by using COS-7
25 cells or CHO cells.
[0242] The transient expression using COS-7 cells was performed as follows. The transfection was performed by
electroporation method using Gene Pulser equipment (BioRad). For the expression of antibody 12B5 (IgG) each 1 0jxg
of the above-mentioned expression vector HEF-12B5H-gy1 and HEF-12 B5L-gK were added, for the expression of
12B5Fab fragment each 1 0jxg of pFd-12B5H and HEF-12B5L-gK were added and for the expression of single chain
30 Fv 1 0jag of pCOS-sc12B5 was added to COS-7 cells (1 X10 7 cells/ml) suspended in 0.8ml of PBS. The mixture kept
in a cuvette was treated by pulse at the capacity of 1 .5kV, 25|^FD. After recovering for 1 0 minutes in a room temperature
the electroporated cells were added to DMEM culture medium (GIBCO BRL) containing 10% bovine fetal serum cul-
tivated. After cultivating overnight the cells were washed once by PBS, added to serum-free medium CHO-S-SFM.II
and cultivated for 2 days. The culture medium was centrifuged to remove cell debris and filtered with 0.22nm filter to
35 prepare the culture supernatant.
[0243] To establish a stable expression CHO cell line for the single chain Fv (polypeptide) derived from antibody
12B5, the expression vector pCHO-sc12B5 was introduced into CHO cells as follows.
[0244] The expression vector was introduced into CHO cells by electroporation method using Gene Pulser equipment
(BioRad). Linearized DNA (1 OOjxg) obtained by digestion with restriction enzyme Pvul and CHO cells (1 x1 0 7 cells /ml)
40 suspended in 0.8 ml of PBS were mixed in a cuvette, left stationary on ice for 1 0 minutes and treated with pulse at the
capacity of 1 .5kV, 25nFD. After recovering for 1 0 minutes at a room temperature the electroporated cells were added
to CHO-S-SFM II (GIBCO BRL) containing 10% bovine fetal serum and cultivated. After cultivating for 2 days the
cultivation was continued in CHO-S-SFM II (GIBCO BRL) containing 5nM methotrexate (SIGMA) and 10% bovine fetal
serum. From thus obtained clones a clone with high expression rate was selected as the production cell line for 1 2B5
45 single chain Fv. After cultivating in serum-free medium CHO-S-SFM II (GIBCO BRL) containing 5nM methotrexate
(SIGMA), the culture supernatant was obtained by centrifugal separation of cell debris.
7.5 Purification of single chain Fv derived from 12B5 produced by CHO cells
so [0245] The culture supernatant of CHO cell line expressing 12B5 single chain Fv obtained in 7.4 was purified by
anti-FLAG antibody column and gel filtration column.
(1 ) Anti-FLAG antibody column
55 [0246] The culture supernatant was added to anti-FLAG M2 affinity gel (SIGMA) equilibrated by PBS. After washing
the column by the same buffer the proteins adsorbed to the column were eluted by 0.1 M glycine-HCI buffer (pH 3.5).
The eluted fractions were immediately neutralized by adding 1M Tris-HCI buffer (pH 8.0). The eluted fractions were
analyzed by SDS-PAGE and the fraction which was confirmed to contain the single chain Fv was concentrated using
29
EP 1 327 681 A1
Centricon-10 (MILLIPORE).
(2) Gel filtration
5 [0247] The concentrated solution obtained in (1) was added to Superdex200 column (10x300mm, AMERSHAM
PHARMACIA) equilibrated by PBS containing 0.01% Tween20.
[0248] The product sc1 2B5 was eluted in two peaks (A, B) (see Fig. 48). The fractions A and B were analyzed using
the 14%-SDS-polyacrylamide gel. The sample was processed by electrophoresis in the presence and absence of a
reducing agent according to Laemmli method, and stained by Coomassie Brilliant Blue after the electrophoresis. As
10 shown in Fig. 49 the fractions A and B, regardless of the presence of the reducing agent or its absence, produced a
single band having an apparent molecular weight of about 31 kD. When the fractions A and B were analyzed by gel
filtration using Superdex200 PC 3.2/30 (3.2x300mm, AMERSHAM PHARMACIA), the fraction A produced an eluted
product at an apparent molecular weight of about 44 kD and the fraction B produced at 22kD (see Fig. 50a and b).
The results show that the fraction A is the non-covalent bond dimer of sc12B5 single chain Fv, and B is the monomer.
15
7.6 Measurement of TPO-like agonist activity of various single chain Fvs
[0249] The TPO-like activity of anti-MPL single chain antibody was evaluated by measuring the proliferation activity
to Ba/F3 cells (BaF/mpI) expressing human TPO receptor (MPL). After washing BaF/MpI cells two times by RPMI1 640
20 culture medium (GIBCO) containing 1 0% bovine fetal serum (GIBCO), the cells were suspended in the culture medium
at cell density of 5x1 0 5 cells/ml. The anti-MPL single chain antibody and human TPO (R&D Systems) was diluted with
the culture medium, respectively. 50^1 of the cell suspension and 50^1 of the diluted antibody or human TPO were
added in 96-well microplate (flat bottom) (Falcon), and cultivated in C0 2 incubator (C0 2 concentration: 5%) for 24
hours. After the incubation 10^1 of WST-8 reagent (reagent for measuring the number of raw cells SF: Nacalai Tesque)
25 was added and the absorbance was immediately measured at measurement wavelength of 450nm and at refference
wavelength of 620nm using fluorescence absorbency photometer SPECTRA Fluor (TECAN). After incubating in C0 2
incubator (C0 2 concentration: 5%) for 2 hours, the absorbance at 450nm of measurement wavelength and 620nm of
refference wavelength was again measured using SPECTRA Fluor. Since WST-8 reagent developed the color reaction
depending upon the number of live cells at wavelength of 450nm, the proliferation activity of BaF/MpI based on the
30 change of absorbance in 2 hours was evaluated by ED 50 calculated as follows. In the proliferation reaction curve
wherein the absorbance was plotted on the ordinate against the antibody concentration on the abscissa, the absorbance
at the plateau was set 100% reaction rate. Obtaining an approximation formula by straight line approximation method
based on the plotted values close to 50% reaction rate, the antibody concentration of 50% reaction rate was calculated
and adopted as ED 50.
35 [0250] The results of the agonist activity to MPL measured by using culture supernatants of COS-7 cells expressing
various 12B5 antibody molecules showed as illustrated in Fig. 51 that 12B5lgG having bivalent antigen-binding site
increased the absorbance in concentration-dependent manner and had TPO-like agonist activity (ED50; 29nM), while
the agonist activity of 1 2B5Fab having monovalent antigen-biding site was very weak (ED50; 34,724nM). On the con-
trary the single chain Fv (sc12B5) having monovalent antigen-binding site like Fab showed strong agonist activity at
40 a level that ED50 was 75nM. However it has been known that variable regions of H chain and L chain of the single
chain Fv are associated through non-covalent bond and, therefore, each variable region is dissociated in a solution
and can be associated with variable region of other molecule to form multimers like dimers. When the molecular weight
of sc12B5 purified by gel filtration was measured, it was confirmed that that there were molecules recognized to be
monomer and dimer (see Fig. 48). Then monomer sc1 2B5 and dimer sc1 2B5 were isolated (see Fig. 50) and measured
45 for the agonist activity to MPL. As shown in Figs. 51 and 52, ED50 of sc12B5 monomer was 4438. 7nM, which confirmed
that the agonist activity was reduced compared with the result using culture supernatant of COS-7 cells. On the contrary
single chain Fv (sc12B5 dimer) having bivalent antigen-binding site showed about 400-fold stronger agonist activity
(ED50; 10.1 nM) compared with monovalent sc12B5. Furthermore, the bivalent single chain Fv showed the agonist
activity equivalent to or higher than the agonist activity of human TPO and 12B5lgG.
50
Example 8
Construction of a gene encoding the variable region of human antibody 12E10 against human MPL
55 [0251] A DNA encoding variable region of human monoclonal antibody 12E1 0 against human MPL was constructed
as follows:
30
EP 1 327 681 A1
8.1 Construction of a gene encoding 12E10 H chain V region
[0252] The nucleotide sequence SEQ ID NO:86 was designed as a gene encoding H chain V region of human
antibody 12E10 binding to human MPL on the basis of the amino acid sequence described in WO99/10494 (SEQ ID
s NO:85). The full length of nucleotide sequence was designed by connecting to its 5'-end the leader sequence (SEQ
ID NO:87) derived from human antibody gene (GenBank accession No. AF062252). The designed nucleotide sequence
was divided into four oligonucleotides having overlapping sequences of 15 bp each (12E10VH1, 12E10VH2,
12E10VH3, 12E10VH4). 12E10VH1 (SEQ ID NO: 88) and 1 2E1 0VH3 (SEQ ID NO: 90) were synthesized in the sense
direction, end 12E10VH2 (SEQ ID NO: 89) end 12E10VH4 (SEQ ID NO: 91) in the antisense direction, respectively.
10 After assembling each synthesized oligonucleotide by respective complementarity, the external primers (12E10VHS
and 12E10VHA) were added to amplify the full length of the gene. 12E10VHS (SEQ ID NO: 92) was designed to
hybridize to 5'-end of the leader sequence by the forward primer and to have Hind III restriction enzyme recognition
site and Kozak sequence, and 12E10VHA (SEQ ID NO: 93) was designed to hybridize to the nucleotide sequence
encoding C-terminal of H chain V region by the reverse primer and to have a splice donor sequence and BamHI
15 restriction enzyme recognition site, respectively.
[0253] 1 00^1 of the PCR solution containing 1 0jil of 1 0 x PCR Gold Buffer II, 1 .5mM MgCI 2 , 0.08mM dNTPs (dATP,
dGTP, dCTP, dTTP), 5 units of DNA-polymerase AmpliTaq Gold (all by PERKIN ELMER) and each 2.5pmole of each
synthesized oligonucleotide (12B5VH-1 to -4) was heated at 94°C of the initial temperature for 9 minutes, at 94°C for
2 minutes, at 55°C for 2 minutes and 72°C for 2 minutes. After repeating the cycle two times each 1 00 pmole of external
20 primer 12E10VHS and 12E10VHA were added. The mixture was subjected to the cycle consisting of at 94°C for 30
seconds, at 55°C for 30 seconds and 72°C for 1 minute 35 times and heated at 72°C for further 5 minutes.
[0254] The PCR product was purified by 1 .5% low-melting-temperature agarose gel (Sigma), digested by restriction
enzymes BamHI and Hind III, and cloned into a human H chain expression vector HEF-gy1 . After determining the DNA
sequence the plasmid containing the correct DNA sequence was named HEF-12E1 0H-gy1 .
25 [0255] The HEF-12E10H-gy1 was digested by restriction enzymes EcoRI and BamHI to produce the gene encoding
12E10VH and then cloned into a human Fab H chain expression vector pCOS-Fd to produce pFd-12E1 OH. The human
Fab H chain expression vector was constructed by amplifying the DNA (SEQ ID NO: 63) containing the intron region
existing between the genes encoding human antibody H chain V region and the constant region, and the gene encoding
a part of the human H chain constant region by PCR, and inserting the PCR product into animal cell expression vector
30 pCOS1 . The human H chain constant region was amplified for the gene under the same conditions mentioned above
using as the template HEF-gy1 , as the forward primer G1 CH1 -S (SEQ ID NO: 64) which was designed to hybridize to
5'-end sequence of intron 1 and to have restriction enzyme recognition sites EcoRI and BamHI and as the reverse
primer G1CH1-A (SEQ ID NO: 65) which was designed to hybridize to 3'-end DNA of human H chain constant region
CH1 domain and to have a sequence encoding a part of hinge region, two stop codons and restriction enzyme recog-
35 nition site Bg1 II.
[0256] The nucleotide sequence and amino acid sequence of the reconstructed 1 2E1 0 H chain variable region which
were included in plasmids HEF-12E10H-gy1 and pFd-12E10H are shown in SEQ ID NO: 94.
8.2 Construction of a gene encoding 12E10 L chain V region
40
[0257] The nucleotide sequence SEQ ID NO:96 was designed as a gene encoding L chain V region of human antibody
12E10 binding to human MPL on the basis of the amino acid sequence described in WO99/10494 (SEQ ID NO:95). It
was further designed by connecting to its 5'-end the leader sequence (SEQ ID NO: 97) derived from human antibody
gene (Mol. Immunol. 1992; 29: 1515-1518). In the same way as mentioned above the designed nucleotide sequence
45 was divided into four oligonucleotides having overlapping sequences of 15 bp each (12E1 0VL1 , 12E1 0VL2, 12E1 0VL3,
12E10VL4) and synthesized respectively. 12E10VL1 (SEQ ID NO: 98) and 12E10VL3 (SEQ ID NO: 100) had sense
sequences, and 12E10VL2 (SEQ ID NO: 99) and 12E10VL4 (SEQ ID NO: 101) had antisense sequences, respectively.
Each of the synthesized oligonucleotides was assembled by respective complementarity and mixed with the external
primers (12E10VLS and 12E10VLA) to amplify the full length of the gene. 12E10VLS (SEQ ID NO: 102) was designed
so to hybridize to 5'-end of the leader sequence by the forward primer and to have EcoRI restriction enzyme recognition
site and Kozak sequence. 12E1 0VLA (SEQ ID NO: 1 03) was designed to hybridize to the nucleotide sequence encoding
C-terminal of L chain V region by the reverse primer and to have a Blnl restriction enzyme recognition site.
[0258] Performing the PCR as mentioned above, the PCR product was purified by 1.5% low-melting-temperature
agarose gel (Sigma), digested by restriction enzymes EcoRI and Blnl, and cloned into pUC19 containing a gene for
55 human lambda chain constant region. After determining the DNA sequence the plasmid containing the correct DNA
sequence was digested by EcoRI to produce a gene encoding 12E10 L chain V region and human lambda chain
constant region and then inserted in expression vector pCOS1 . The plasmid having 12E10 L chain gene (SEQ ID NO:
1 04) was named pCOS-1 2E1 0L
31
EP 1 327 681 A1
8.3 Production of reconstructed 12E10 single chain Fv
[0259] The reconstructed 12E10 antibody single chain Fv was designed to be in the order of 12E10VH-linker-
12E10VL and to have FLAG sequence (SEQ ID NO: 105) at C-terminal to facilitate the detection and purification. The
s reconstructed 12E10 chain Fvs (sc12E10 and db12E10) were constructed using a linker sequence consisting of 15
amino acids represented by (Gly 4 Ser) 3 or 5 amino acids represented by (Gly 4 Ser) 1 .
(1) Production of the reconstructed 12E10 single chain Fv using the linker sequence consisting of 5 amino acids
10 [0260] The gene encoding the reconstructed 1 2E1 0 single chain Fv, which contained the linker sequence consisting
of 5 amino acids, was constructed by introducing the nucleotide sequence for the linker (Gly 4 Ser) 1 to 3'-end of the
gene encoding 12E10 H chain V region and to 5'-end of the gene encoding 12E10 L chain V region, amplifying thus
obtained respective gene by PCR and connecting the amplified genes. Four PCR primers (A-D) were used to produce
the reconstructed 1 2E1 0 single chain Fv. Primers A and C had sense sequences, and primers B and D had antisense
15 sequences.
[0261] The forward primer for H chain V region was 12E10S (Primer A, SEQ ID NO: 106). The reverse primer DB2
(Primer B, SEQ ID NO: 1 07) for H chain V region was designed to hybridize to DNA encoding C-terminal of H chain V
region and to have the nucleotide sequence encoding the linker (Gly 4 Ser).| and the nucleotide sequence encoding N-
terminal of L chain V region.
20 [0262] The forward primer DB1 (Primer C, SEQ ID NO: 1 08) for L chain V region was designed to hybridize to DNA
encoding the N-terminal of L chain V region and to have the nucleotide sequence encoding the linker (Gly 4 Ser) 1 and
the nucleotide sequence encoding C-terminal of H chain V region. The reverse primer 12E10FA (Primer D, SEQ ID
NO: 109) for L chain V region was designed to hybridize to DNA encoding the C-terminal of L chain V region and to
have the nucleotide sequence encoding FLAG and Notl restriction enzyme recognition site.
25 [0263] In the first PCR step, two reactions A-B and C-D were performed, and the two PCR products obtained from
the first step PCR were assembled by respective complementarity. After adding primers A and D the full length DNA
encoding the reconstructed 12E10 single chain Fv having the linker consisting of 5 amino acids was amplified (the
second PCR). In the first step PCR, the plasmid HEF-12E10H-gy1 (see Example 8. 1) encoding the reconstructed
12E10 H chain V region and pCOS-12E10L (see Example 8.1) encoding the reconstructed 12E10 L chain V region
30 were used as templates, respectively.
[0264] 50|il of the first step PCR solution contained 5^1 of 10 x PCR Gold Buffer II, 1.5mM MgCI 2 , 0.08mM dNTPs,
5 units of DNA polymerase AmpliTaq Gold (by PERKIN ELMER), each 100 pmole of each primer and 100ng of each
template DNA. The PCR solution was heated at 94°C of the initial temperature for 9 minutes, at 94 for 30 seconds,
55°C for 30 seconds and 72°C for 1 minute. After repeating the cycle 35 times the reaction mixture was further heated
35 at 72°C for 5 minutes.
[0265] The PCR products A-B (429bp) and C-D (395bp) were assembled by the second PCR. The second step PCR
mixture solution (98jxl) containing 1jxl each of the first PCR product A-B and C-D as templates, 1 00 pmole each of each
primer, 10^1 of 10 x PCR Gold Buffer II, 1 .5mM MgCI 2 , 0.08mM dNTPs and 5 units of DNA polymerase AmpliTaq Gold
(by PERKIN ELMER) was reacted under the same conditions as mentioned above.
40 [0266] The DNA fragment of 795bp produced by the second PCR was purified using 1 .5% low-melting-temperature
agarose gel, digested by EcoRI and Notl, and cloned into pCHOI vector or pCOS1 vector. The expression vector
pCHOI was a vector constructed by deleting the antibody gene from DHFR-AE-RVH-PM1-f (see W092/19759) by
EcoRI and Smal digestion, and connecting to EcoRI-Notl-BamHI Adaptor (TAKARA SHUZO). After determining the
DNA sequence the plasmids containing the DNA fragment encoding the correct amino acid sequence of reconstructed
45 12B5 single chain Fv were named pCHO-db12E10 and pCOS-db12E10. The nucleotide sequence and amino acid
sequence of the reconstructed 1 2E1 0 single chain Fv included in the plasmids pCHO-db1 2E1 0 and pCOS-db1 2-E1 0
are shown in SEQ ID NO: 110.
(2) Production of the reconstructed 12E10 single chain Fv using the linker sequence consisting of 15 amino acids
50
[0267] The gene encoding the reconstructed 12E10 antibody single chain Fv, which contained the linker sequence
consisting of 15 amino acids, was constructed by introducing the nucleotide sequence for the linker (Gly 4 Ser) 3 to 3'-
end of the gene encoding 12E1 0 H chain V region and to 5'-endof the gene encoding 12E1 0 L chain V region, amplifying
thus obtained respective gene by PCR and connecting the amplified genes. Four PCR primers (A-D) were used for
55 production of the reconstructed 12E10 single chain Fv. Primers A and C had sense sequences, and primers B and D
had antisense sequences.
[0268] The forward primer for H chain V region was 12E10S (Primer A, SEQ ID NO: 106). The reverse primer sc4.3
(Primer B, SEQ ID NO: 111) for H chain V region was designed to hybridize to DNA encoding C-terminal of H chain V
32
EP 1 327 681 A1
region and to have the nucleotide sequence encoding the linker (Gly 4 Ser) 3 and the nucleotide sequence encoding N-
terminal of L chain V region.
[0269] The forward primer sc1 .3 (Primer C, SEQ ID NO: 112) for L chain V region was designed to hybridize to DNA
encoding the N-terminal of L chain V region and to have the nucleotide sequence encoding the linker (Gly 4 Ser) 3 and
s the nucleotide sequence encoding C-terminal of H chain V region. The reverse primer 12E10FA (Primer D, SEQ ID
NO: 109) for L chain V region was designed to hybridize to DNA encoding the C-terminal of L chain V region and to
have the nucleotide sequence encoding FLAG and Notl restriction enzyme recognition site.
[0270] In the first PCR step, two reactions A-B and C-D were performed, and the two PCR products obtained from
the first step PCR were assembled by respective complementarity. After adding primers A and D the full length DNA
10 encoding the reconstructed 12E10 single chain Fv having the linker consisting of 15 amino acids was amplified (the
second PCR). In the first step PCR, the plasmid pCOS-db12E10 (see Example 8. 1(1)) encoding the reconstructed
12E10 single chain Fv was used as template.
[0271 ] 50^1 of the first step PCR solution contained 5|xl of 1 0 x ExTaq Buffer, 0.4mM dNTPs, 2.5 units of DNA polymer-
ase TaKaRa ExTaq (by TAKARA), each 1 00 pmole of each primer and 1 0ng of each template DNA. The PCR solution
15 was heated at 94°C of the initial temperature for 30 seconds, at 94 for 15 seconds and 72°C for 2 minute, and the
cycle was repeated 5 times. After repeating 28 times the cycle of at 94°C for 15 seconds and at 70°C for 2 minutes,
the reaction mixture was further heated at 72°C for 5 minutes.
[0272] The PCR products A-B (477bp) and C-D (447bp) were assembled by the second PCR. The second step PCR
mixture solution (98jj,I) containing 1 each of the first PCR products A-B and C-D as templates, 100 pmole each of
20 each primer A and D, 5nlof 10x ExTaq Buffer, 0.4mM dNTPs, 2.5 units of DNA polymerase TaKaRa ExTaq (by TAKARA)
was reacted under the same conditions as mentioned above.
[0273] The DNA fragment of 825bp produced by the second PCR was purified using 1 .0% low-melting-temperature
agarose gel, digested by EcoRI and Notl. Thus obtained DNA fragment was cloned into pCHOI vector or pCOS1
vector. After determining the DNA sequence the plasmids containing the DNA fragment encoding the correct amino
25 acid sequence of reconstructed 1 2E1 0 single chain Fv were named pCHO-sc1 2E1 0 and pCOS-sc1 2E1 0. The nucle-
otide sequence and amino acid sequence of the reconstructed 1 2E1 0 single chain Fv included in the plasmids pCHO-
SC12E10 and pCOS-sc12E10 are shown in SEQ ID NO: 113.
8.4 Expression of antibody 12E10 (IgG, Fab) and single chain Fv polypeptide by animal cell
30
[0274] Antibody 1 2E1 0 (IgG, Fab) and single chain Fv derived from antibody 1 2E1 0 (linker sequence 5 amino acids,
15 amino acids) were expressed by using COS-7 cells or CHO cells.
[0275] The transient expression using COS-7 cells was performed as follows. The transfection was performed by
electroporation method using Gene Pulser II equipment (BioRad). For the expression of antibody 12E10 (IgG) each
35 10^g of the above-mentioned expression vector HEF-12E1 0H-gy1 and pCOS-1 2E1 0L were added, for the expression
of 12E10Fab fragment each 1 0jxg of pFd-12E10H and pCOS-12E10L were added and for the expression of single
chain Fv of pCOS-sc12E10 (1 0|j,g) or pCOS-db12E10 (10jig) was added to COS-7 cells (1 X10 7 cells/ml) suspended
in 0.8ml of PBS. The mixture kept in a cuvette was treated by pulse at the capacity of 1 .5kV, 25jxFD. After recovering
for 1 0 minutes in a room temperature the electroporated cells were added to DMEM medium (GIBCO BRL) containing
40 1 o% bovine fetal serum and cultivated. After cultivating overnight the cells were washed once by PBS, added to serum-
free medium CHO-S-SFM II (GIBCO BRL) and cultivated for 3 days. The culture supernatant was centrifuged to remove
cell debris and filtered with 0.22nm filter.
[0276] To establish a stable expression CHO cell line for the single chain Fv (polypeptide) derived from antibody
12E10, the expression vector pCHO-sc12E10 or pCHO-ds12E10 was introduced into CHO cells respectively.
45 [0277] Each expression vectorwas introduced into CHO cells by electroporation method using Gene Pulser II equip-
ment (BioRad). Linearized DNA (1 OO^ig) obtained by digestion with restriction enzyme Pvul and CHO cells (1x1 0 7 cells
/ml) suspended in 0.8 ml of PBS were mixed in a cuvette, left stationary on ice for 10 minutes and treated with pulse
at the capacity of 1 .5kV, 25jxFD. After recovering for 1 0 minutes at a room temperature the electroporated cells were
added to CHO-S-SFM II medium (GIBCO BRL) containing 10% dialyzed bovine fetal serum and nucleic acid and
so cultivated. After cultivating for 2 days the cultivation was continued in nucleic acid-free CHO-S-SFM II medium (GIBCO
BRL) containing 10% dialyzed bovine fetal serum. From thus obtained clones a clone with high expression rate was
selected as the production cell line for 12E10 single chain Fv. After cultivating in serum-free CHO-S-SFM II medium
(GIBCO BRL), the culture supernatant was centrifuged to remove cell debris and filtered with 0.22nm filter.
55 8.5 Purification of single chain Fv derived from 1 2E1 0 produced by CHO cells
[0278] The culture supernatants produced by CHO cell lines expressing 1 2E1 0 single chain Fvs (sc1 2E1 0, db1 2E1 0)
obtained in Example 8.4 were purified by anti-FLAG antibody column and gel filtration column respectively to produce
33
EP 1 327 681 A1
purified single chain Fvs.
(1) Purification with anti-FLAG antibody column
s [0279] Each culture supernatant (sc12E10db12E10) was added to anti-FLAG M2 affinity gel column (SIGMA) equil-
ibrated by 50mM Tris-HCI buffer (pH7.4) containing 150mM NaCI. After washing the column by the same buffer the
proteins adsorbed to the column were eluted by 1 0OmM glycine buffer (pH 3.5). The eluted fractions were immediately
neutralized by adding 1 M Tris-HCI buffer (pH 8.0) and analyzed by SDS-PAGE. The fraction which was confirmed to
contain the single chain Fv was pooled and concentrated about 20-fold using Centricon-10 (AMICON).
10
(2) Gel filtration
[0280] The concentrated solution obtained in (1) was added to Superdex200 column HR (10x300mm, AMERSHAM
PHARMACIA) equilibrated by PBS containing 0.01% Tween20. Chlomatograms were shown in Fig. 53 and 54. The
15 product sc12E10 was eluted in two peaks (A, B) (see Fig. 53). The product db12E10 was eluted in two peaks (C, D)
(see Fig. 54). Each peak fraction was collected, treated in the presence and absence of a reducing agent, processed
by electrophoresis according to Laemmli method and stained by Coomassie Brilliant Blue after the electrophoresis.
As shown in Fig. 55 the all of fractions A, B, C and D, regardless of the presence or absence of the reducing agent,
produced a single band having an apparent molecular weight of about 31 kD. When these fractions were analyzed by
20 gel filtration using Superdex200 HR, the fraction A produced a product eluted at an apparent molecular weight of about
20 kD, the fraction B at 42kD (see Fig. 56), fraction C at 69kD and fraction D at 41 kD (see Fig. 57). The results suggest
that sc12E1 0-derive fraction A is the non-covalent bond dimer of single chain Fv and the fraction B is the monomer of
single chain Fv, and the db12E10-derived fraction C is the non-covalent bond trimerof single chain Fv and D is non-
covalent bond dimer of single chain Fv.
25
8.6 Measurement of TPO-like agonist activity of various single chain Fvs
[0281] The TPO-like activity of anti-mpl single chain antibody was evaluated by measuring the proliferation activity
to Ba/F3 cells (BaF/mpI) expressing human TPO receptor (MPL).
30 [0282] After washing BaF/mpI cells two times by RPMI1640 medium (GIBCO) containing 1% bovine fetal serum
(GIBCO), the cells were suspended in the medium at cell density of 5x1 0 5 cells/mL. The anti-M PL single chain antibody
or human TPO (R&D Systems) was diluted with the medium, respectively. 50^1 of the cell suspension and 50^1 of the
diluted antibody or human TPO were added in 96-well microplate (flat bottom) (Corning), and cultivated in C0 2 incubator
(C0 2 concentration: 5%) for 24 hours. After the incubation 1 OjliI of WST-8 reagent (reagent for measuring the number
35 of raw cells SF: Nacalai Tesque) was added and the absorbance was immediately measured at measurement wave-
length of 450nm and at reference wavelength of 655nm using absorbency photometer Benchmark Plus (BioRad). After
incubating in C0 2 incubator (C0 2 concentration: 5%) for 2 hours, the absorbance at 450nm of measurement wavelength
and 655nm of reference wavelength was again measured using Benchmark Plus. Since WST-8 reagent developed
the color reaction depending upon the number of live cells at wavelength of 450nm, the proliferation activity of BaF/
40 mpl was evaluated based on the change of absorbance in 2 hours.
[0283] The agonist activity to MPL measured by using culture supernatants of COS-7 cells expressing various 12E1 0
antibody molecules are shown in Fig. 58. Single chain Fvs having the 5-amino-acid-linker (ds12E1 0 and the 15-amino-
acid-linker (sc12E1 0) increased the absorbance in concentration-dependent manner, showing TPO-like agonist activity
(ED50; 9pM and 51 pM respectively), while 12E10lgG and 12E10Fab had no activity.
45 [0284] It has been known that H chain and L chain of the single chain Fv are associated not only within a molecule
but also between molecules to form multimers such as dimer. When the culture supernatants of CHO cells expressing
single chain Fvs of 12E1 0 were gel filtrated and tested for agonist activity on MPL. The results were shown in Fig. 59.
The dimer, which was contained in sc12E10 in a small amount, showed about 5000-fold stronger TPO-like agonist
activity (sc12E1 0 dimer, ED50; 1 .9pM) compared with the monomer (sc12E10 monomer, ED50; >1 OnM). The activity
so was higher than that of TPO (ED50; 27pM). The dimer of db12E10 (db12E10 dimer, ED50;2.0pM) showed strong
activity comparable to that of sc12E1 0 dimer. db12E1 0 trimer (ED50; 7.4pM), which was presumed to be a trimer from
molecular weight obtained by gel filtration, showed a high activity which is lower than that of db12E10 dimer. Those
results suggest that it is important for the activity of agonist antibody 12E10 that the antigen-binding site is bivalent
(dimer). Considering the fact that 12E10 IgG had no activity, other factors than being bivalent are presumed to be
55 important such as the Location of antigen-binding site, the distance or the angle.
34
EP 1 327 681 A1
EXPLANATION OF DRAWINGS
[0285]
s Fig. 1 shows the result of flow cytometry, illustrating that human IgG antibody does not bind to L1210 cells ex-
pressing human IAP (MAP/L1210).
Fig. 2 shows the result of flow cytometry, illustrating that the chimera MABL-1 antibody specifically binds to L121 0
cells expressing human IAP (hlAP/L1210).
Fig. 3 shows the result of flow cytometry, illustrating that the chimera MABL-2 antibody specifically binds to L121 0
10 cells expressing human IAP (hlAP/L1210).
Fig. 4 schematically illustrates the process for producing the single chain Fv according to the present invention.
Fig. 5 illustrates a structure of an expression plasmid which can be used to express a DNA encoding the single
chain Fv of the invention in E. coli.
Fig. 6 illustrates a structure of an expression plasmid which is used to express a DNA encoding the single chain
15 Fv of the invention in mammalian cells.
Fig. 7 shows the result of western blotting in Example 5.4. From the left, a molecular weight marker (which indicates
97.4, 66, 45, 31 , 21 .5 and 1 4.5 kDa from the top), the culture supernatant of pCHOI -introduced COS7 cells and
the culture supernatant of pCHOM2-introduced COS7 cells. It illustrates that the reconstructed single chain Fv of
the antibody MABL-2 (arrow) is contained in the culture supernatant of the pCHOM2-introduced cells.
20 Fig. 8 shows the result of flow cytometry, illustrating that an antibody in the culture supernatant of pCH01/COS7
cell as a control does not bind to pCOS1/L1210 cell as a control.
Fig. 9 shows the result of flow cytometry, illustrating that an antibody in the culture supernatant of MABL2-scFv/
COS7 cells does not bind to pCOS1 /L1 21 0 cells as a control.
Fig. 1 0 shows the result of flow cytometry, illustrating that an antibody in the culture supernatant of pCOS1/COS7
25 cells as a control does not bind to MAP/L121 0 cells.
Fig. 11 shows the result of flow cytometry, illustrating that an antibody in the culture supernatant of MABL2-scFv/
COS7 cells specifically binds to MAP/L1210 cells.
Fig. 12 shows the result of the competitive ELISA in Example 5.6, wherein the binding activity of the single chain
Fv of the invention (MABL2-scFv) to the antigen is demonstrated in terms of the inhibition of binding of the mouse
30 monoclonal antibody MABL-2 to the antigen as an index, in comparison with the culture supernatant of
pCH01/COS7 cells as a control.
Fig. 13 shows the results of the apoptosis-inducing effect in Example 5.7, illustrating that the antibody in the culture
supernatant of pCH01/COS7 cells as a control does not induce the apoptosis of pCOS1/L121 0 cells as a control.
Fig. 14 shows the results of the apoptosis-inducing effect in Example 5.7, illustrating that the antibody in the culture
35 supernatant of MABL2-scFv/COS7 cells does not induce apoptosis of pCOS1/L1210 cells as acontrol.
Fig. 15 shows the results of the apoptosis-inducing effect in Example 5.7, illustrating that the antibody in the culture
supernatant of pCH01/COS7 cells as acontrol does not induce apoptosis of hlAP/L1210 cells.
Fig. 1 6 shows the results of the apoptosis-inducing effect in Example 5.7, illustrating that the antibody in the culture
supernatant of MABL2-scFv/COS7 cells specifically induces apoptosis of hlAP/L1210 cells.
40 Fig. 1 7 shows the results of the apoptosis-inducing effect in Example 5.7, illustrating that the antibody in the culture
supernatant of pCH01/COS7 cells as a control does not induce apoptosis of CCRF-CEM cells (at 50% of the final
concentration).
Fig. 1 8 shows the results of the apoptosis-inducing effect in Example 5.7, illustrating that the antibody in the culture
supernatant of MABL2-scFv/COS7 cells specifically induces apoptosis of CCRF-CEM cells (at 50% of the final
45 concentration).
Fig. 1 9 shows the chromatogram obtained in the purification of the single chain Fv derived form the antibody MABL-
2 produced by the CHO cells in Example 5.9, illustrating that fraction A and fraction B were obtained as the major
peaks when the fraction from Blue-sepharose column was purified with hydroxyapatite column.
Fig. 20 shows the results of purification by gel filtration of fraction A and fraction B obtained in Example 5.9-(2),
so illustrating that the major peaks (Al and Bl, respectively) were eluted from fraction A at approximately 36 kD of the
apparent molecular weight and from fraction B at approximately 76 kD.
Fig. 21 is the analysis on SDS-PAGE of the fractions obtained in the purification of the single chain Fv derived
from the antibody MABL-2 produced by the CHO cells in Example 5.9, illustrating that a single band of approxi-
mately 35 kD of molecular weight was observed in both fractions.
55 Fig. 22 shows the results of analysis of fractions Al and Bl obtained by gel filtration in the purification of the single
chain Fv derived from the antibody MABL-2 produced by the CHO cells, wherein fraction Al comprises monomer
and fraction Bl comprises dimer.
Fig. 23 illustrates a structure of an expression plasmid which can be used to express a DNA encoding the single
35
EP 1 327 681 A1
chain Fv of the invention in E. coli.
Fig. 24 shows the results of purification on the gel filtration column of crude products of the single chain Fv polypep-
tide derived from the antibody MABL-2 produced by E. coli obtained in Example 5.12, wherein each peak indicates
monomer or dimer, respectively, of the single chain Fv produced by E. coli.
s Fig. 25 shows the results of the apoptosis-inducing effect in Example 5.13, illustrating that mouse IgG antibody as
a control does not induce apoptosis of hlAP/L1210 cells (the final concentration of 3|^g/ml).
Fig. 26 shows the results of the apoptosis-inducing effect in Example 5.13, illustrating that the dimer of MABL2-scFv
produced by the CHO cells remarkably induces apoptosis of hlAP/L121 0 cells (the final concentration of 3 ng/ml).
Fig. 27 shows the results of the apoptosis-inducing effect in Example 5.13, illustrating that the dimer of MABL2-scFv
10 produced by E^ coli remarkably induces apoptosis of hlAP/L121 0 cells (the final concentration of 3 ng/ml).
Fig. 28 shows the results of the apoptosis-inducing effect in Example 5.13, illustrating that apoptosis induction to
hlAP/L121 0 cells by the MABL2-scFv monomer produced by the CHO cells is the same level as that of the control
(the final concentration of 3 ng/ml).
Fig. 29 shows the results of the apoptosis-inducing effect in Example 5.13, illustrating that apoptosis induction to
15 hlAP/L121 0 cells of the MABL2-scFv monomer produced by E. coli is the same level as that of control (the final
concentration of 3 |ag/ml).
Fig. 30 shows the results of the apoptosis-inducing effect in Example 5.13, illustrating that mouse IgG antibody
used as a control does not induce apoptosis of hlAP/L121 0 cells even when anti-FLAG antibody is added (the final
concentration of 3 |ag/ml).
20 Fig. 31 shows the results of the apoptosis-inducing effect in Example 5.13, illustrating that MABL2-scFv monomer
produced by the CHO cells remarkably induces apoptosis of hlAP/L1 21 0 cells when anti-FLAG antibody is added
(the final concentration of 3 ng/ml).
Fig. 32 shows the results of quantitative measurement of human IgG in the serum of a human myeloma cell line
KPMM2-transplanted mouse, indicating amounts of human IgG produced by the human myeloma cells in the
25 mouse. It illustrates that the dimer of scFv/CHO remarkably inhibited growth of the KPMM2 cells.
Fig. 33 shows the survival time of the mouse after the transplantation of tumor, illustrating that the scFv/CHO
dimer-administered group elongated remarkably the survival time.
Fig. 34 illustrates a structure of an expression plasmid which expresses a modified antibody [sc(Fv) 2 ] comprising
two H chain V regions and two L chain V regions derived from the antibody MABL-2.
30 Fig. 35 illustrates a structure of a plasmid which expresses a scFv (HL type) wherein the V regions are linked in
the manner of [H chain]-[L chain] without a peptide linker.
Fig. 36 illustrates a structure of the HL-type polypeptide and amino acid sequences of peptide linkers.
Fig. 37 illustrates a structure of a plasmid which expresses a scFv (LH type) wherein the V regions are linked in
the manner of [L chain]-[H chain] without a peptide linker.
35 Fig. 38 illustrates a structure of the LH-type polypeptide and amino acid sequences of peptide linkers.
Fig. 39 shows the results of the western blotting in Example 6.4, illustrating that the modified antibody sc(FV) 2
comprising two H chain V regions and two L chain V regions, and the MABL2-scFv having peptide linkers with
different length are expressed.
Figs. 40a and 40b show the results of flow cytometry using the culture supernatant of COS7 cells prepared in
40 Example 6.3 (1), illustrating that the MABL2-scFv and sc(Fv) 2 having peptide linkers with different length have
high affinities against human IAP.
Fig. 41 shows the results of the apoptosis-inducing effect in Example 6.6, illustrating that the scFv <HL3, 4, 6, 7,
LH3, 4, 6 and 7> and the sc(Fv) 2 remarkably induce cell death of hlAP/L1210 cells.
Fig. 42 shows the results of the evaluation of antigen binding capacity in Example 6.1 0, illustrating that the dimer
45 of scFv <HL5> and sc(Fv) 2 have high affinities against human IAP.
Fig. 43 shows the results of the in vitro apoptosis-inducing effect in Example 6.11, illustrating that the dimer of
scFv <HL5> and the sc(Fv) 2 induce apoptosis of hlAP/L121 0 cells and CCRF-CEM cells in concentration-depend-
ent manner.
Fig. 44 shows the results of the quantitative measurement of M protein produced by a human myeloma cell line
so KPMM2 in the serum of the human myeloma cell-transplanted mouse. It illustrates that the dimer of scFv <HL5>
and the sc(Fv) 2 remarkably inhibited growth of the KPMM2 cells.
Fig. 45 shows the survival time (days) of mice after the transplantation of tumor, illustrating that the survival time
of the scFv <HL5> administrated-group was remarkably prolonged.
Fig. 46 shows the survival time (days) of mice after the transplantation of tumor, illustrating that the survival time
55 of the sc(Fv) 2 administrated-group was remarkably prolonged.
Fig. 47 is a scheme showing the method for constructing DNA fragment encoding the reconstructed 12B5 single
chain Fv containing the linker sequence consisting of 15 amino acids and the structure thereof.
Fig. 48 shows the purification result of each 12B5 single chain Fv by gel filtration obtained in Example 7. 5 (1),
36
EP 1 327 681 A1
illustrating that sc12B5 was divided into two peaks (fractions A and B).
Fig. 49 shows the analytical result of each fraction A and B by SDS-PAGE performed in Example 7. 5 (2).
Fig. 50 shows the analytical result of each fraction A and B by Superdex200 column performed in Example 7. 5
(2), illustrating that the major peak of fraction A was eluted at an apparent molecular weight of about 44 kD shown
s in (a) and that the major peak of fraction B was eluted at an apparent molecular weight of about 22kD shown in (b).
Fig. 51 shows the measurement result of the TPO-like agonist activity of sc12B5 and antibody 12B5 (IgG, Fab),
illustrating that 12B5lgG and monovalent single chain Fv (sc12B5) showed TPO-like agonist activity in concentra-
tion-dependent manner.
Fig. 52 shows the measurement result of TOP-like agonist activity of sc12B5 monomer and dimer, illustrating that
10 single chain Fv (sc12B5 dimer) having bivalent antigen-binding site had agonist activity about 400-fold higher than
monovalent sc12B5 and that the efficacy is equivalent to or higher than human TPO.
Fig. 53 shows the purification result of obtained sc12E10 single chain antibody by gel filtration chromatography
using Superdex200HR column, illustrating that 12E10sc3 was divided into two peaks (fractions A and B).
Fig. 54 shows the purification result of obtained db12E10 single chain antibody by gel filtration chromatography
15 using Superdex200HR column, illustrating that 12E10sc3 was divided into two peaks (fractions C and D).
Fig. 55. shows SDS-PAGE analysis of fractions A and B (sc12E10) and fractions C and D (db12E10) under the
reductive or non-reductive condition.
Fig. 56 shows the analytical result of fractions A and B by gel filtration chromatography using Superdex200HR
column, illustrating (1) the major peak of fraction A was eluted at an apparent molecular weight of about 42 kD
20 and (2) the major peak of fraction B was eluted at an apparent molecular weight of about 20kD.
Fig. 57 shows the analytical result of fractions C and D by gel filtration chromatography using Superdex200HR
column, illustrating (1) the major peak of fraction C was eluted at an apparent molecular weight of about 69 kD
and (2) the major peak of fraction B was eluted at an apparent molecular weight of about 41 kD.
Fig. 58 is a graph showing the agonist activity of various 12E1 0 antibody molecules on MPL, illustrating that single
25 chain Fvs (sc12E10, db12E10) showed TPO-like agonist activity while 12E10 IgG and 12E10 Fab did not.
Fig. 59 is a graph showing the agonist activity of monomer and dimer of sc12E1 0 and dimer and trimer of db12E1 0
on MPL, illustrating that dimer of sc12E10 and dimer and trimer of db12E10 showed TPO-like agonist activity
higher than TPO.
30 INDUSTRIAL APPLICABILITY
[0286] The modified antibodies of the invention have an agonist action capable of transducing a signal into cells by
crosslinking a cell surface molecule(s) and are advantageous in that the permeability to tissues and tumors is high due
to the lowered molecular size compared with the parent antibody molecule (whole IgG). The present invention provides
35 the modified antibodies which have remarkably high agonist activity compared with natural ligands such as TPO and
the parent antibody (whole IgG). Even if the parent antibody has no agonist activity, modified antibodies with a higher
agonist activity compared with natural ligands can be provided. This is attributable to that the modified antibodies are
in a shape closer to a ligand as compared with original antibodies. Therefore the modified antibodies can be used as
signal-transducing agonists to achieve apoptosis induction, cell proliferation induction, cell differentiation induction,
40 cell division induction or cell cycle regulation action. The modification of antibody molecule to the modified antibody
according to the invention results in the reduction of side effects caused by intercellular crosslinking and provides novel
medicines inducing only required action by crosslinking a cell surface molecule(s). Medical preparations containing as
active ingredient the modified antibody of the invention are useful as preventives and/or remedies for cancers, inflam-
mation, hormone disorders, autoimmune diseases and blood diseases, for example, leukemia, malignant lymphoma,
45 aplastic anemia, myelodysplasia syndrome and polycythemia vera.
37
EP 1 327 681 A1
SEQUENCE LISTING
<110> CHUGAI SEIYAKU KABUSHIKI KAISHA
<120> Small remodeling agonist antibody
<130> FP1032
<141> 2001-10-22
<150> JP2000-321821
<151> 2000-10-20
<150> JP2000-321822
<151> 2000-10-20
<150> PCT/JP01/01912
<151> 2001-03-12
<150> PCT/JPO 1/03288
<151> 2001-04-17
<150> JP2001-277314
<151> 2001-09-12
<160> 113
<210> 1
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> PCR primer
<400> 1
ccatcctaat acgactcact atagggc 27
38
EP 1 327 681 A1
<210> 2
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> PCR primer
<400> 2
ggatcccggg tggatggtgg gaagatg 27
<210> 3
<211> 28
<212> DNA
<213> Artificial Sequence
<220>
<223> PCR primer
<400> 3
ggatcccggg ccagtggata gacagatg 28
<210> 4
<211> 26
<212> DNA
<213> Artificial Sequence
<220>
<223> PCR primer
<400> 4
ggatcccggg agtggataga ccgatg 26
<210> 5
39
EP 1 327 681 A1
10
15
<211> 394
<212> DNA
<213> Mus
<220>
<221> CDS
<222> (1)... (393)
<223> pGEM-MlL. 1-57; signal peptide, 58-394;mature peptide
<400> 5
atg aag ttg cct gtt agg ctg ttg gtg ctg atg ttc tgg att cct gcg 48
20 Met Lys Leu Pro Val Arg Leu Leu Val Leu Met Phe Trp lie Pro Ala
1 5 10 15
tec age agt gat gtt gtg atg ace caa act cca etc tec ctg cct gtc 96
25
Ser Ser Ser Asp Val Val Met Thr Gin Thr Pro Leu Ser Leu Pro Val
20 25 30
30 agt ctt gga gat caa gec tec ate tct tgc aga tct agt cag age ctt 144
Ser Leu Gly Asp Gin Ala Ser lie Ser Cys Arg Ser Ser Gin Ser Leu
35 40 45
eta cac agt aaa gga aac ace tat tta caa tgg tac eta cag aag cca 192
Leu His Ser Lys Gly Asn Thr Tyr Leu Gin Trp Tyr Leu Gin Lys Pro
50 55 60
ggc cag tct cca aag etc ctg ate tac aaa gtt tec aac cga ttt tct 240
Gly Gin Ser Pro Lys Leu Leu lie Tyr Lys Val Ser Asn Arg Phe Ser
45 65 70 75 80
ggg gtc cca gac agg ttc agt ggc agt gga tea ggg aca gat ttc aca 288
Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr
85 90 95
etc aag ate age aga gtg gag get gag gat ctg gga gtt tat ttc tgc 336
Leu Lys He Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys
35
40
50
40
EP 1 327 681 A1
10
15
40
45
100 105 110
tct caa agt aca cat gtt ccg tac acg tec gga ggg ggg acc aag ctg 384
Ser Gin Ser Thr His Val Pro Tyr Thr Ser Gly Gly Gly Thr Lys Leu
115 120 125
gaa ata aaa c 394
Glu He Lys
130
<210> 6
20 <211> 409
<212> DNA
<213> Mus
25
<220>
<221> CDS
<222> (1)... (408)
30
<223> pGEM-MlH. 1-57 signal peptide, 58-409 Mature peptide
<400> 6
35 atg gaa tgg age tgg ata ttt etc ttc etc ctg tea gga act gca ggt 48
Met Glu Trp Ser Trp lie Phe Leu Phe Leu Leu Ser Gly Thr Ala Gly
15 10 15
gtc cac tec cag gtc cag ctg cag cag tct gga cct gac ctg gta aag 96
Val His Ser Gin Val Gin Leu Gin Gin Ser Gly Pro Asp Leu Val Lys
20 25 30
cct ggg get tea gtg aag atg tec tgc aag get tct gga tac acc ttc 144
Pro Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe
so 35 40 45
gtt aac cat gtt atg cac tgg gtg aag cag aag cca ggg cag ggc ctt 192
Val Asn His Val Met His Trp Val Lys Gin Lys Pro Gly Gin Gly Leu
41
EP 1 327 681 A1
10
15
50 55 60
gag tgg att gga tat att tat cct tac aat gat ggt act aag tac aat 240
Glu Trp He Gly Tyr lie Tyr Pro Tyr Asn Asp Gly Thr Lys Tyr Asn
65 70 75 80
gag aag ttc aag ggc aag gcc aca ctg act tea gag aaa tec tec age 288
Glu Lys Phe Lys Gly Lys Ala Thr Leu Thr Ser Glu Lys Ser Ser Ser
85 90 95
gca gcc tac atg gag etc age age ctg gcc tct gag gac tct gcg gtc 336
Ala Ala Tyr Met Glu Leu Ser Ser Leu Ala Ser Glu Asp Ser Ala Val
20 .. 100 105 110
tac tac tgt gca aga ggg ggt tac tat agt tac gac gac tgg ggc caa 384
Tyr Tyr Cys Ala Arg Gly Gly Tyr Tyr Ser Tyr Asp Asp Trp Gly Gin
115 120 125
ggc acc act etc aca gtc tec tea g 409
Gly Thr Thr Leu Thr Val Ser Ser
30
130 135
<210> 7
<211> 394
<212> DNA
<213> Mus
<220>
<221> CDS
<222> (1)... (393)
<223> pGEM-M2L. l-57;signal peptide, 58-394;mature peptide
<400> 7
atg aag ttg cct gtt agg ctg ttg gtg ctg atg ttc tgg att cct ggt 48
Met Lys Leu Pro Val Arg Leu Leu Val Leu Met Phe Trp lie Pro Gly
42
10
15
EP 1 327 681 A1
1 5 10 15
tec age agt gat gtt gtg atg acc caa agt cca etc tec ctg cct gtc 96
Ser Ser Ser Asp Val Val Met Thr Gin Ser Pro Leu Ser Leu Pro Val
20 25 30
agt ctt gga gat caa gee tec ate tct tgc aga tea agt cag age ctt 144
Ser Leu Gly Asp Gin Ala Ser lie Ser Cys Arg Ser Ser Gin Ser Leu
35 40 45
gtg cac agt aat gga aag acc tat tta cat tgg tac ctg cag aag cca 192
Val His Ser Asn Gly Lys Thr Tyr Leu His Trp Tyr Leu Gin Lys Pro
20 , 50 55 60
ggc cag tct cca aaa etc ctg ate tac aaa gtt tec aac cga ttt tct 240
Gly Gin Ser Pro Lys Leu Leu He Tyr Lys Val Ser Asn Arg Phe Ser
65 70 75 80
ggg gtc cca g ac a SS ttc a e* gg c a gt g§ a tca gtg aca ttc aca 288
3o Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Val Thr Asp Phe Thr
85 90 95
etc atg ate age aga gtg gag get gag gat ctg gga gtt tat ttc tgc 336
35 Leu Met He Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys
100 105 110
tct caa agt aca cat gtt ccg tac acg ttc gga ggg ggg acc aag ctg 384
Ser Gin Ser Thr His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu
115 120 125
gaa ata aaa c 394
Glu He Lys
130
40
45
50
<210> 8
<211> 409
43
EP 1 327 681 A1
<212> DNA
<213> Mus
<220>
<221> CDS
<222> (1). . . (408)
<223> pGEM-M2H. 1-57; signal peptide, 58-409 Jmature peptide
<400> 8
atg gaa tgg age tgg ata ttt etc ttc etc ctg tea gga act gca ggt
Met Glu Trp Ser Trp lie Phe Leu Phe Leu Leu Ser Gly Thr Ala Gly
-15 10 15
gtc cac tec cag gtc cag ctg cag cag tct gga cct gaa ctg gta aag
Val His Ser Gin Val Gin Leu Gin Gin Ser Gly Pro Glu Leu Val Lys
20 25 30
cct ggg get tea gtg aag atg tec tgc aag get tct gga tac acc ttc
Pro Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe
35 40 45
get aac cat gtt att cac tgg gtg aag cag aag cca ggg cag ggc ctt
Ala Asn His Val He His Trp Val Lys Gin Lys Pro Gly Gin Gly Leu
50 55 60
gag tgg att gga tat att tat cct tac aat gat ggt act aag tat aat
Glu Trp He Gly Tyr lie Tyr Pro Tyr Asn Asp Gly Thr Lys Tyr Asn
65 70 75 80
gag aag ttc aag gac aag gec act ctg act tea gac aaa tec tec acc
Glu Lys Phe Lys Asp Lys Ala Thr Leu Thr Ser Asp Lys Ser Ser Thr
85 90 95
aca gee tac atg gac etc age age ctg gee tct gag gac tct gcg gtc
Thr Ala Tyr Met Asp Leu Ser Ser Leu Ala Ser Glu Asp Ser Ala Val
100 105 110
44
EP 1 327 681 A1
tat tac tgt gca aga ggg ggt tac tat act tac gac gac tgg ggc caa 384
5 Tyr Tyr Cys Ala Arg Gly Gly Tyr Tyr Thr Tyr Asp Asp Trp Gly Gin
115 120 125
ggc acc act etc aca gtc tec tea g 409
10
Gly Thr Thr Leu Thr Val Ser Ser
130 135
15
<210> 9
<211> 32
20 <212> DNA
<213> Artificial Sequence
<220>
25
<223> PCR primer
<400> 9
3Q cccaagcttc caccatgaag ttgcctgtta gg 32
<210> 10
35 <211> 32
<212> DNA
<213> Artificial Sequence
40
<220>
<223> PCR primer
<400> 10
45
cccaagcttc caccatggaa tggagctgga ta 32
so <210> 11
<211> 34
<212> DNA
45
EP 1 327 681 A1
<213> Artificial Sequence
<220>
<223> PCR primer
<400> 11
cgcggatcca ctcacgtttt atttccagct tggt 34
<210> 12
<211> 34
<212> DNA
<213> Artificial Sequence
<220>
<223> PCR primer
<400> 12
cgcggatcca ctcacctgag gagactgtga gagt 34
<210> 13
<211> 30
<212> DNA
<213> Artificial Sequence
<220>
<223> PCR primer
<400> 13
catgccatgg cgcaggtcca gctgcagcag 30
<210> 14
<211> 27
<212> DNA
<213> Artificial Sequence
46
EP 1 327 681 A1
<22Q>
s <223> PCR primer
<400> 14
accaccacct gaggagactg tgagagt 27
10
<210> 15
<211> 27
15
<212> DNA
<213> Artificial Sequence
20 <220>
<223> PCR primer
<400> 15
25
gtctcctcag gtggtggtgg ttcgggt 27
<210> 16
30
<211> 27
<212> DNA
35 <213> Artificial Sequence
<220>
<223> PCR primer
40 <400> 16
cacaacatcc gatccgccac cacccga 27
45
<210> 17
<211> 27
so <212> DNA
<213> Artificial Sequence
<220>
47
EP 1 327 681 A1
<223> PCR primer
<400> 17
ggcggatcgg atgttgtgat gacccaa 27
<210> 18
<211> 57
<212> DNA
<213> Artificial Sequence
<220>
<223> PCR primer
<400> 18
ccggaattct cattatttat cgtcatcgtc tttgtagtct tttatttcca gcttggt 57
<210> 19
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Linker amino acid sequence and nucleotide sequence
<400> 19
ggt ggt ggt ggt teg ggt ggt ggt ggt teg ggt ggt ggc gga teg 45
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
5 10 15
<210> 20
<211> 828
<212> DNA
<213> Mus
48
EP 1 327 681 A1
10
15
<220>
<221> CDS
<222> (1). . . (822)
<223> pscMl. MABLl-scFv
<400> 20
atg aaa tac eta ttg cct acg gca gec get gga ttg tta tta etc get 48
Met Lys Tyr Leu Leu Pro Thr Ala Ala Ala Gly Leu Leu Leu Leu Ala
15 10 15
gec caa cca gec atg gcg cag gtc cag ctg cag cag tct gga cct gac 96
20 Ala Gin Pro Ala Met Ala Gin Val Gin Leu Gin Gin Ser Gly Pro Asp
20 25 30
ctg gta aag cct ggg get tea gtg aag atg tec tgc aag get tct gga 144
25
Leu Val Lys Pro Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly
35 40 45
tac acc ttc gtt aac cat gtt atg cac tgg gtg aag cag aag cca ggg 192
30
Tyr Thr Phe Val Asn His Val Met His Trp Val Lys Gin Lys Pro Gly
50 55 60
cag ggc ctt gag tgg att gga tat att tat cct tac aat gat ggt act 240
Gin Gly Leu Glu Trp He Gly Tyr He Tyr Pro Tyr Asn Asp Gly Thr
65 70 75 80
aag tac aat gag aag ttc aag ggc aag gec aca ctg act tea gag aaa 288
Lys Tyr Asn Glu Lys Phe Lys Gly Lys Ala Thr Leu Thr Ser Glu Lys
85 90 95
tec tec age gca gec tac atg gag etc age age ctg gec tct gag gac 336
Ser Ser Ser Ala Ala Tyr Met Glu Leu Ser Ser Leu Ala Ser Glu Asp
100 105 110
tct gcg gtc tac tac tgt gca aga ggg ggt tac tat agt tac gac gac 384
Ser Ala Val Tyr Tyr Cys Ala Arg Gly Gly Tyr Tyr Ser Tyr Asp Asp
35
40
45
50
49
EP 1 327 681 A1
10
15
20
115 120 125
tgg ggc caa ggc acc act etc aca gtc tec tea ggt ggt ggt ggt teg 432
Trp Gly Gin Gly Thr Thr Leu Thr Val Ser Ser Gly Gly Gly Gly Ser
130 135 140
ggt ggt ggt ggt teg ggt ggt ggc gga teg gat gtt gtg atg acc caa 480
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Val Val Met Thr Gin
145 150 155 160
act cca etc tec ctg cct gtc agt ctt gga gat caa gee tec ate tct 528
Thr Pro Leu Ser Leu Pro Val Ser Leu Gly Asp Gin Ala Ser He Ser
165 170 175
tgc aga tct agt cag age ctt eta cac agt aaa gga aac acc tat tta 576
Cys Arg Ser Ser Gin Ser Leu Leu His Ser Lys Gly Asn Thr Tyr Leu
25
180 185 190
caa tgg tac eta cag aag cca ggc cag tct cca aag etc ctg ate tac 624
30 Gin Trp Tyr Leu Gin Lys Pro Gly Gin Ser Pro Lys Leu Leu He Tyr
195 200 205
aaa gtt tec aac cga ttt tct ggg gtc cca gac agg ttc agt ggc agt 672
35 Lys Val Ser Asn Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser
210 215 220
gga tea ggg aca gat ttc aca etc aag ate age aga gtg gag get gag 720
Gly Ser Gly Thr Asp Phe Thr Leu Lys He Ser Arg Val Glu Ala Glu
225 230 235 240
gat ctg gga gtt tat ttc tgc tct caa agt aca cat gtt ccg tac acg 768
Asp Leu Gly Val Tyr Phe Cys Ser Gin Ser Thr His Val Pro Tyr Thr
245 250 255
so tec gga ggg ggg acc aag ctg gaa ata aaa gac tac aaa gac gat gac 816
Ser Gly Gly Gly Thr Lys Leu Glu He Lys Asp Tyr Lys Asp Asp Asp
260 265 270
40
45
50
EP 1 327 681 A1
gat aaa taatga 828
5 Asp Lys
<210> 21
10
<211> 31
<212> DNA
15 <213> Artificial Sequence
<220>
<223> PCR primer
20 <400> 21
acgcgtcgac tcccaggtcc agctgcagca g 31
<210> 22
<211> 18
<212> DNA
30
<213> Artificial Sequence
<220>
as <223> PCR primer
<400> 22
gaaggtgtat ccagaagc 18
40
<210> 23
<211> 819
45
<212> DNA
<213> Mus
so <220>
<221> CDS
<222> (1). . . (813)
51
EP 1 327 681 A1
10
15
<223> pCHOMl. MABLl-scFv
<400> 23
atg gga tgg age tgt ate ate etc ttc ttg gta gca aca get aca ggt 48
Met Gly Trp Ser Cys lie He Leu Phe Leu Val Ala Thr Ala Thr Gly
15 10 15
gtc gac tec cag gtc cag ctg cag cag tct gga cct gac ctg gta aag 96
Val Asp Ser Gin Val Gin Leu Gin Gin Ser Gly Pro Asp Leu Val Lys
20 25 30
cct ggg get tea gtg aag atg tec tgc aag get tct gga tac acc ttc 144
20 Pro Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe
35 40 45
gtt aac cat gtt atg cac tgg gtg aag cag aag cca ggg cag ggc ctt 192
Val Asn His Val Met His Trp Val Lys Gin Lys Pro Gly Gin Gly Leu
50 55 60
gag tgg att gga tat att tat cct tac aat gat ggt act aag tac aat 240
30
Glu Trp lie Gly Tyr lie Tyr Pro Tyr Asn Asp Gly Thr Lys Tyr Asn
65 70 75 80
gag aag ttc aag ggc aag gec aca ctg act tea gag aaa tec tec age 288
Glu Lys Phe Lys Gly Lys Ala Thr Leu Thr Ser Glu Lys Ser Ser Ser
85 90 95
gca gec tac atg gag etc age age ctg gee tct gag gac tct gcg gtc 336
Ala Ala Tyr Met Glu Leu Ser Ser Leu Ala Ser Glu Asp Ser Ala Val
100 105 110
tac tac tgt gca aga ggg ggt tac tat agt tac gac gac tgg ggc caa 384
Tyr Tyr Cys Ala Arg Gly Gly Tyr Tyr Ser Tyr Asp Asp Trp Gly Gin
115 120 125
ggc acc act etc aca gtc tec tea ggt ggt ggt ggt teg ggt ggt ggt 432
Gly Thr Thr Leu Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly
35
40
45
50
52
EP 1 327 681 A1
130 135 140
ggt teg ggt ggt ggc gga teg gat gtt gtg atg acc caa act cca etc 480
Gly Ser Gly Gly Gly Gly Ser Asp Val Val Met Thr Gin Thr Pro Leu
145 150 155 160
tec ctg cct gtc agt ctt gga gat caa gec tec ate tct tgc aga tct 528
Ser Leu Pro Val Ser Leu Gly Asp Gin Ala Ser He Ser Cys Arg Ser
165 170 175
agt cag age ctt eta cac agt aaa gga aac acc tat tta caa tgg tac 576
Ser Gin Ser Leu Leu His Ser Lys Gly Asn Thr Tyr Leu Gin Trp Tyr
180 185 190
eta cag aag cca ggc cag tct cca aag etc ctg ate tac aaa gtt tec 624
Leu Gin Lys Pro Gly Gin Ser Pro Lys Leu Leu lie Tyr Lys Val Ser
195 200 205
aac cga ttt tct ggg gtc cca gac agg ttc agt ggc agt gga tea ggg 672
Asn Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly
210 215 220
aca gat ttc aca etc aag ate age aga gtg gag get gag gat ctg gga 720
Thr Asp Phe Thr Leu Lys lie Ser Arg Val Glu Ala Glu Asp Leu Gly
225 230 235 240
gtt tat ttc tgc tct caa agt aca cat gtt ccg tac acg tec gga ggg 768
Val Tyr Phe Cys Ser Gin Ser Thr His Val Pro Tyr Thr Ser Gly Gly
245 250 255
ggg acc aag ctg gaa ata aaa gac tac aaa gac gat gac gat aaa taa 816
Gly Thr Lys Leu Glu He Lys Asp Tyr Lys Asp Asp Asp Asp Lys
260 265 270
tga 819
<210> 24
53
EP 1 327 681 A1
10
15
<211> 828
<212> DNA
<213> Mus
<220>
<221> CDS
<222> (1). . . (822)
<223> pscM2. MABL2-scFv
<400> 24
atg aaa tac eta ttg cct acg gca gec get gga ttg tta tta etc get 48
20 Met Lys Tyr Leu Leu Pro Thr Ala Ala Ala Gly Leu Leu Leu Leu Ala
1 5 10 15
gec caa cca gec atg gcg cag gtc cag ctg cag cag tct gga cct gaa 96
Ala Gin Pro Ala Met Ala Gin Val Gin Leu Gin Gin Ser Gly Pro Glu
20 25 30
ctg gta aag cct ggg get tea gtg aag atg tec tgc aag get tct gga 144
30
Leu Val Lys Pro Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly
35 40 45
tac ace ttc get aac cat gtt att cac tgg gtg aag cag aag cca ggg 192
Tyr Thr Phe Ala Asn His Val lie His Trp Val Lys Gin Lys Pro Gly
50 55 60
cag ggc ctt gag tgg att gga tat att tat cct tac aat gat ggt act 240
Gin Gly Leu Glu Trp He Gly Tyr lie Tyr Pro Tyr Asn Asp Gly Thr
65 70 75 80
aag tat aat gag aag ttc aag gac aag gec act ctg act tea gac aaa 288
Lys Tyr Asn Glu Lys Phe Lys Asp Lys Ala Thr Leu Thr Ser Asp Lys
85 90 95
tec tec acc aca gee tac atg gac etc age age ctg gec tct gag gac 336
Ser Ser Thr Thr Ala Tyr Met Asp Leu Ser Ser Leu Ala Ser Glu Asp
35
40
45
50
54
EP 1 327 681 A1
100 105 110
tct gcg gtc tat tac tgt gca aga ggg ggt tac tat act tac gac gac 384
Ser Ala Val Tyr Tyr Cys Ala Arg Gly Gly Tyr Tyr Thr Tyr Asp Asp
115 120 125
tgg ggc caa ggc acc act etc aca gtc tec tea ggt ggt ggt ggt teg 432
Trp Gly Gin Gly Thr Thr Leu Thr Val Ser Ser Gly Gly Gly Gly Ser
130 135 140
ggt ggt ggt ggt teg ggt ggt ggc gga teg gat gtt gtg atg acc caa 480
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Val Val Met Thr Gin
L45 150 155 160
agt cca etc tec ctg cct gtc agt ctt gga gat caa gee tec ate tct 528
Ser Pro Leu Ser Leu Pro Val Ser Leu Gly Asp Gin Ala Ser He Ser
165 170 175
tgc aga tea agt cag age ctt gtg cac agt aat gga aag acc tat tta 576
Cys Arg Ser Ser Gin Ser Leu Val His Ser Asn Gly Lys Thr Tyr
180 185 190
cat tgg tac ctg cag aag cca ggc cag tct cca aaa etc ctg ate tac 624
His Trp Tyr Leu Gin Lys Pro Gly Gin Ser Pro Lys Leu Leu He Tyr
195 200 205
aaa gtt tec aac cga ttt tct ggg gtc cca gac agg ttc agt ggc agt 672
Lys Val Ser Asn Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser
210 215 220
gga tea gtg aca gat ttc aca etc atg ate age aga gtg gag get gag 720
Gly Ser Val Thr Asp Phe Thr Leu Met lie Ser Arg Val Glu Ala Glu
225 230 235 240
gat ctg gga gtt tat ttc tgc tct caa agt aca cat gtt ccg tac acg 768
Asp Leu Gly Val Tyr Phe Cys Ser Gin Ser Thr His Val Pro Tyr Thr
245 250 255
55
EP 1 327 681 A1
ttc gga ggg ggg acc aag ctg gaa ata aaa gac tac aaa gac gat gac 816
5 Phe Gly Gly Gly Thr Lys Leu Glu lie Lys Asp Tyr Lys Asp Asp Asp
260 265 270 .
gat aaa taatga 828
10
Asp Lys
<210> 25
15
<211> 819
<212> DNA
20 <213> Mus
<220>
<221> CDS
<222> (1).. . (813)
<223> pCH0M2. MABL2-scFv
<400> 25
30
atg gga tgg age tgt ate ate etc ttc ttg gta gca aca get aca ggt 48
Met Gly Trp Ser Cys lie lie Leu Phe Leu Val Ala Thr Ala Thr Gly
35 1 5 10 15
gtc gac tec cag gtc cag ctg cag cag tct gga cct gaa ctg gta aag 96
Val Asp Ser Gin Val Gin Leu Gin Gin Ser Gly Pro Glu Leu Val Lys
20 25 30
cct ggg get tea gtg aag atg tec tgc aag get tct gga tac acc ttc 144
Pro Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe
35 40 45
get aac cat gtt att cac tgg gtg aag cag aag cca ggg cag ggc ctt 192
so Ala Asn His Val He His Trp Val Lys Gin Lys Pro Gly Gin Gly Leu
50 55 60
gag tgg att gga tat att tat cct tac aat gat ggt act aag tat aat 240
40
45
56
EP 1 327 681 A1
10
15
Glu Trp He Gly Tyr He Tyr Pro Tyr Asn Asp Gly Thr Lys Tyr Asn
65 70 75 80
gag aag ttc aag gac aag gcc act ctg act tea gac aaa tec tec acc 288
Glu Lys Phe Lys Asp Lys Ala Thr Leu Thr Ser Asp Lys Ser Ser Thr
85 90 95
aca gcc tac atg gac etc age age ctg gcc tct gag gac tct gcg gtc 336
Thr Ala Tyr Met Asp Leu Ser Ser Leu Ala Ser Glu Asp Ser Ala Val
100 105 110
tat tac tgt gca aga ggg ggt tac tat act tac gac gac tgg ggc caa 384
20 Tyr Tyr Cys Ala Arg Gly Gly Tyr Tyr Thr Tyr Asp Asp Trp Gly Gin
115 120 125
ggc acc act etc aca gtc tec tea ggt ggt ggt ggt teg ggt ggt ggt 432
Gly Thr Thr Leu Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly
130 135 140
ggt teg ggt ggt ggc gga teg gat gtt gtg atg acc caa agt cca etc 480
30
Gly Ser Gly Gly Gly Gly Ser Asp Val Val Met Thr Gin Ser Pro Leu
145 150 155 160
35 tec ctg cct gtc agt ctt gga gat caa gcc tec ate tct tgc aga tea 528
Ser Leu Pro Val Ser Leu Gly Asp Gin Ala Ser He Ser Cys Arg Ser
165 170 175
agt cag age ctt gtg cac agt aat gga aag acc tat tta cat tgg tac 576
Ser Gin Ser Leu Val His Ser Asn Gly Lys Thr Tyr Leu His Trp Tyr
180 185 190
ctg cag aag cca ggc cag tct cca aaa etc ctg ate tac aaa gtt tec 624
Leu Gin Lys Pro Gly Gin Ser Pro Lys Leu Leu He Tyr Lys Val Ser
195 200 205
aac cga ttt tct ggg gtc cca gac agg ttc agt ggc agt gga tea gtg 672
Asn Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Val
40
45
50
57
EP 1 327 681 A1
10
15
210 215 220
aca gat ttc aca etc atg ate age aga gtg gag get gag gat ctg gga 720
Thr Asp Phe Thr Leu Met He Ser Arg Val Glu Ala Glu Asp Leu Gly
225 230 235 240
gtt tat ttc tgc tct caa agt aca cat gtt ccg tac acg ttc gga ggg 768
Val Tyr Phe Cys Ser Gin Ser Thr His Val Pro Tyr Thr Phe Gly Gly
245 250 255
ggg acc aag ctg gaa ata aaa gac tac aaa gac gat gac gat aaa taa 816
Gly Thr Lys Leu Glu lie Lys Asp Tyr Lys Asp Asp Asp Asp Lys
20 v 260 265 270
tga 819
<210> 26
<211> 456
<212> DNA
30
<213> Mus
<220>
35 <221> CDS
<222> (1).. . (450)
<223> pCHO-shlAP. Soluble human IAP
<400> 26
atg tgg ccc ctg gta gcg gcg ctg ttg ctg ggc teg gcg tgc tgc gga 48
Met Trp Pro Leu Val Ala Ala Leu Leu Leu Gly Ser Ala Cys Cys Gly
15 10 15
tea get cag eta eta ttt aat aaa aca aaa tct gta gaa ttc acg ttt 96
so Ser Ala Gin Leu Leu Phe Asn Lys Thr Lys Ser Val Glu Phe Thr Phe
20 25 30
tgt aat gac act gtc gtc att cca tgc ttt gtt act aat atg gag gca 144
40
45
58
EP 1 327 681 A1
10
15
Cys Asn Asp Thr Val Val lie Pro Cys Phe Val Thr Asn Met Glu Ala
35 40 45
caa aac act act gaa gta tac gta aag tgg aaa ttt aaa gga aga gat 192
Gin Asn Thr Thr Glu Val Tyr Val Lys Trp Lys Phe Lys Gly Arg Asp
50 55 60
att tac acc ttt gat gga get eta aac aag tec act gtc ccc act gac 240
He Tyr Thr Phe Asp Gly Ala Leu Asn Lys Ser Thr Val Pro Thr Asp
65 70 75 80
ttt agt agt gca aaa att gaa gtc tea caa tta eta aaa gga gat gec 288
20 Phe Ser Ser Ala Lys He Glu Val Ser Gin Leu Leu Lys Gly Asp Ala
85 90 95
tct ttg aag atg gat aag agt gat get gtc tea cac aca gga aac tac 336
25
Ser Leu Lys Met Asp Lys Ser Asp Ala Val Ser His Thr Gly Asn Tyr
100 105 110
3Q act tgt gaa gta aca gaa tta acc aga gaa ggt gaa acg ate ate gag 384
Thr Cys Glu Val Thr Glu Leu Thr Arg Glu Gly Glu Thr lie He Glu
115 120 125
35 eta aaa tat cgt gtt gtt tea tgg ttt tct cca aat gaa aat gac tac 432
Leu Lys Tyr Arg Val Val Ser Trp Phe Ser Pro Asn Glu Asn Asp Tyr
130 135 140
40
aag gac gac gat gac aag tgatag 456
Lys Asp Asp Asp Asp Lys
145 150
45
<210> 27
so <211> 46
<212> DNA
<213> Artificial Sequence
59
EP 1 327 681 A1
<220>
<223> PCR primer
<400> 27
ggaattccat atgcaagtgc aacttcaaca gtctggacct gaactg 46
<210> 28
<211> 31
<212> DNA
<213> Artificial Sequence
<220>
<223> PCR primer
<400> 28
ggaattctca ttattttatt tccagcttgg t 31
<210> 29
<211> 741
<212> DNA
<213> Mus
<220>
<221> CDS
<222> (1). . . (735)
<223> pscM2DEra02. MABL2-scFv
<400> 29
atg caa gtg caa ctt caa cag tct gga cct gaa ctg gta aag cct ggg
Met Gin Val Gin Leu Gin Gin Ser Gly Pro Glu Leu Val Lys Pro Gly
15 10 15
get tea gtg aag atg tec tgc aag get tct gga tac ace ttc get aac
Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ala Asn
60
EP 1 327 681 A1
10
15
20 25 30
cat gtt att cac tgg gtg aag cag aag cca ggg cag ggc ctt gag tgg 144
His Val He His Trp Val Lys Gin Lys Pro Gly Gin Gly Leu Glu Trp
35 40 45
att gga tat att tat cct tac aat gat ggt act aag tat aat gag aag 192
lie Gly Tyr lie Tyr Pro Tyr Asn Asp Gly Thr Lys Tyr Asn Glu Lys
50 55 60
ttc aag gac aag gcc act ctg act tea gac aaa tec tec acc aca gec 240
Phe Lys Asp Lys Ala Thr Leu Thr Ser Asp Lys Ser Ser Thr Thr Ala
20 .65 70 75 80
tac atg gac etc age age ctg gcc tct gag gac tct gcg gtc tat tac 288
Tyr Met Asp Leu Ser Ser Leu Ala Ser Glu Asp Ser Ala Val Tyr Tyr
25 85 90 95
tgt gca aga ggg ggt tac tat act tac gac gac tgg ggc caa ggc acc 336
Cys Ala Arg Gly Gly Tyr Tyr Thr Tyr Asp Asp Trp Gly Gin Gly Thr
30
100 105 110
act etc aca gtc tec tea ggt ggt ggt ggt teg ggt ggt ggt ggt teg 384
35 Thr Leu Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
115 120 125
ggt ggt ggc gga teg gat gtt gtg atg acc caa agt cca etc tec ctg 432
Gly Gly Gly Gly Ser Asp Val Val Met Thr Gin Ser Pro Leu Ser Leu
130 135 140
cct gtc agt ctt gga gat caa gcc tec ate tct tgc aga tea agt cag 480
Pro Val Ser Leu Gly Asp Gin Ala Ser He Ser Cys Arg Ser Ser Gin
145 150 155 ' 160
age ctt gtg cac agt aat gga aag acc tat tta cat tgg tac ctg cag 528
Ser Leu Val His Ser Asn Gly Lys Thr Tyr Leu His Trp Tyr Leu Gin
165 170 175
40
45
50
61
EP 1 327 681 A1
10
15
aag cca ggc cag tct cca aaa etc ctg ate tac aaa gtt tec aac cga 576
Lys Pro Gly Gin Ser Pro Lys Leu Leu He Tyr Lys Val Ser Asn Arg
180 185 190
ttt tct ggg gtc cca gac agg ttc agt ggc agt gga tea gtg aca gat 624
Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Val Thr Asp
195 200 205
ttc aca etc atg ate age aga gtg gag get gag gat ctg gga gtt tat 672
Phe Thr Leu Met He Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr
210 215 220
20 ttc tgc tct caa agt aca cat gtt ccg tac acg ttc gga ggg ggg acc 720
Phe Cys Ser Gin Ser Thr His Val Pro Tyr Thr Phe Gly Gly Gly Thr
225 230 235 240
25
aag ctg gaa ata aaa taatga 741
Lys Leu Glu lie Lys
245
<210> 30
35 <211> 18
<212> DNA
<213> Artificial Sequence
40 <220>
<223> PCR primer
<400> 30
45
cagacagtgg ttcaaagt 18
<210> 31
<211> 72
<212> DNA
62
EP 1 327 681 A1
<213> Artificial Sequence
<220>
<223> PCR primer
<400> 31
cgcgtcgacc gatccgccac cacccgaacc accaccaccc gaaccaccac caccttttat 60
ttccagcttg gt 72
<210> 32
<211> 1605
<212> DNA
<213> Mus
<220>
<221> CDS
<222> (1).. . (1599)
<223> pCH0M2(Fv)2. MABL2-sc (Fv) 2
<400> 32
atg gga tgg age tgt ate ate etc ttc ttg gta gca aca get aca ggt 48
Met Gly Trp Ser Cys lie He Leu Phe Leu Val Ala Thr Ala Thr Gly
15 10 15
gtc gac tec cag gtc cag ctg cag cag tct gga cct gaa ctg gta aag 96
Val Asp Ser Gin Val Gin Leu Gin Gin Ser Gly Pro Glu Leu Val Lys
20 25 30
cct ggg get tea gtg aag atg tec tgc aag get tct gga tac acc ttc 144
Pro Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe
35 40 45
get aac cat gtt att cac tgg gtg aag cag aag cca ggg cag ggc ctt 192
Ala Asn His Val He His Trp Val Lys Gin Lys Pro Gly Gin Gly Leu
50 55 . 60
63
EP 1 327 681 A1
10
15
gag tgg att gga tat att tat cct tac aat gat ggt act aag tat aat 240
Glu Trp lie Gly Tyr lie Tyr Pro Tyr Asn Asp Gly Thr Lys Tyr Asn
65 70 75 80
gag aag ttc aag gac aag gcc act ctg act tea gac aaa tec tec acc 288
Glu Lys Phe Lys Asp Lys Ala Thr Leu Thr Ser Asp Lys Ser Ser Thr
85 90 95
aca gcc tac atg gac etc age age ctg gcc tct gag gac tct gcg gtc 336
Thr Ala Tyr Met Asp Leu Ser Ser Leu Ala Ser Glu Asp Ser Ala Val
100 105 110
20 tat tac tgt gca aga ggg ggt tac tat act tac gac gac tgg ggc caa 384
Tyr Tyr Cys Ala Arg Gly Gly Tyr Tyr Thr Tyr Asp Asp Trp Gly Gin
115 120 125
25
ggc acc act etc aca gtc tec tea ggt ggt ggt ggt teg ggt ggt ggt 432
Gly Thr Thr Leu Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly
130 135 140
30
ggt teg ggt ggt ggc gga teg gat gtt gtg atg acc caa agt cca etc 480
Gly Ser Gly Gly Gly Gly Ser Asp Val Val Met Thr Gin Ser Pro Leu
ss 145 150 155 160
tec ctg cct gtc agt ctt gga gat caa gcc tec ate tct tgc aga tea 528
Ser Leu Pro Val Ser Leu Gly Asp Gin Ala Ser lie Ser Cys Arg Ser
165 170 175
agt cag age ctt gtg cac agt aat gga aag acc tat tta cat tgg tac 576
Ser Gin Ser Leu Val His Ser Asn Gly Lys Thr Tyr Leu His Trp Tyr
180 185 190
ctg cag aag cca ggc cag tct cca aaa etc ctg ate tac aaa gtt tec 624
so Leu Gin Lys Pro Gly Gin Ser Pro Lys Leu Leu lie Tyr Lys Val Ser
195 200 205
aac cga ttt tct ggg gtc cca gac agg ttc agt ggc agt gga tea gtg 672
40
45
64
EP 1 327 681 A1
10
20
Asn Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Val
210 215 220
aca gat ttc aca etc atg ate age aga gtg gag get gag gat ctg gga 720
Thr Asp Phe Thr Leu Met He Ser Arg Val Glu Ala Glu Asp Leu Gly
225 230 235 240
gtt tat ttc tgc tct caa agt aca cat gtt ccg tac acg ttc gga ggg 768
15 Val Tyr Phe Cys Ser Gin Ser Thr His Val Pro Tyr Thr Phe Gly Gly
245 250 255
ggg acc aag ctg gaa ata aaa ggt ggt ggt ggt teg ggt ggt ggt ggt 816
Giy Thr Lys Leu Glu lie Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly
260 265 270
teg ggt ggt ggc gga teg gtc gac tec cag gtc cag ctg cag cag tct 864
25
Ser Gly Gly Gly Gly Ser Val Asp Ser Gin Val Gin Leu Gin Gin Ser
275 280 285
30 gga cct gaa ctg gta aag cct ggg get tea gtg aag atg tec tgc aag 912
Gly Pro Glu Leu Val Lys Pro Gly Ala Ser Val Lys Met Ser Cys Lys
290 295 300
35 get tct gga tac acc ttc get aac cat gtt att cac tgg gtg aag cag 960
Ala Ser Gly Tyr Thr Phe Ala Asn His Val He His Trp Val Lys Gin
305 310 315 320
aag cca ggg cag ggc ctt gag tgg att gga tat att tat cct tac aat 1008
Lys Pro Gly Gin Gly Leu Glu Trp He Gly Tyr He Tyr Pro Tyr Asn
325 330 335
gat ggt act aag tat aat gag aag ttc aag gac aag gec act ctg act 1056
Asp Gly Thr Lys Tyr Asn Glu Lys Phe Lys Asp Lys Ala Thr Leu Thr
so 340 345 350
tea gac aaa tec tec acc aca gee tac atg gac etc age age ctg gee 1104
Ser Asp Lys Ser Ser Thr Thr Ala Tyr Met Asp Leu Ser Ser Leu Ala
40
45
65
EP 1 327 681 A1
355 360 365
tct gag gac tct gcg gtc tat tac tgt gca aga ggg ggt tac tat act 1152
Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg Gly Gly Tyr Tyr Thr
370 375 380
tac gac gac tgg ggc caa ggc acc act etc aca gtc tec tea ggt ggt 1200
Tyr Asp Asp Trp Gly Gin Gly Thr Thr Leu Thr Val Ser Ser Gly Gly
385 390 395 400
ggt ggt teg ggt ggt ggt ggt teg ggt ggt ggc gga teg gat gtt gtg 1248
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Val Val
405 410 415
atg acc caa agt cca etc tec ctg cct gtc agt ctt gga gat caa gec 1296
Met Thr Gin Ser Pro Leu Ser Leu Pro Val Ser Leu Gly Asp Gin Ala
420 425 430
tec ate tct tgc aga tea agt cag age ctt gtg cac agt aat gga aag 1344
Ser lie Ser Cys Arg Ser Ser Gin Ser Leu Val His Ser Asn Gly Lys
435 440 445
acc tat tta cat tgg tac ctg cag aag cca ggc cag tct cca aaa etc 1392
Thr Tyr Leu His Trp Tyr Leu Gin Lys Pro Gly Gin Ser Pro Lys Leu
450 455 460
ctg ate tac aaa gtt tec aac cga ttt tct ggg gtc cca gac agg ttc 1440
Leu lie Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro Asp Arg Phe
465 470 475 480
agt ggc agt gga tea gtg aca gat ttc aca etc atg ate age aga gtg 1488
Ser Gly Ser Gly Ser Val Thr Asp Phe Thr Leu Met He Ser Arg Val
485 490 495
gag get gag gat ctg gga gtt tat ttc tgc tct caa agt aca cat gtt 1536
Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser Gin Ser Thr His Val
500 505 510
66
EP 1 327 681 A1
ccg tac acg ttc gga ggg ggg acc aag ctg gaa ata aaa gac tac aaa
Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu He Lys Asp Tyr Lys
515 520 525
gac gat gac gat aaa taatga
Asp Asp Asp Asp Lys
530
<210> 33
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> PCR primer
<400> 33
tgaggaattc ccaccatggg atg 33
<210> 34
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> PCR primer
<400> 34
cacgacgtca ctcgagactg tgagagtggt gccttggccc 40
<210> 35
<211> 40
<212> DNA
67
EP 1 327 681 A1
<213> Artificial Sequence
<220>
<223> PCR primer
<400> 35
agtctcgagt gacgtcgtga tgacccaaag tccactctcc
<210> 36
<211> 31
<212> DNA
<213> Artificial Sequence
<220>
<223> PCR primer
<400> 36
gactggatcc tcattattta tcgtcatcgt c 31
<210> 37
<211> 22
<212> DNA
<213> Artificial Sequence
<220>
<223> PCR primer
<400> 37
cgcgtaatac gactcactat ag 22
<210> 38
<211> 46
<212> DNA
<213> Artificial Sequence
68
EP 1 327 681 A1
<220>
<223> PCR primer
<400> 38
gcaattggac ctgttttatc tcgagcttgg tcccccctcc gaacgt 46
<210> 39
<211> 45
<212> DNA
<213> Artificial Sequence
<:220>
<223> PCR primer
<400> 39
gctcgagata aaacaggtcc aattgcagca gtctggacct gaact 45
<210> 40
<211> 60
<212> DNA
<213> Artificial Sequence
<220>
<223> PCR primer
<400> 40
gactggatcc tcattattta tcgtcatcgt ctttgtagtc tgaggagact gtgagagtgg
<210> 41
<211> 32
<212> DNA
<213> Artificial Sequence
<220>
69
EP 1 327 681 A1
<223> PCR primer
5 <400> 41
gactgaattc ccaccatgaa gttgcctgtt ag 32
10
<210> 42
<211> 40
is <212> DNA
<213> Artificial Sequence
<220>
20 <223> PCR primer
<400> 42
cagtctcgag tggtggttcc gacgtc'gtga tgacccaaag 40
25
<210> 43
30 <211> 43
<212> DNA
<213> Artificial Sequence
35 <220>
<223> PCR primer
<400> 43
40
cagtctcgag tggtggtggt tccgacgtcg tgatgaccca aag 43
<210> 44
<211> 46
<212> DNA
<213> Artificial Sequence
<220>
<223> PCR primer
70
EP 1 327 681 A1
<400> 44
cagtctcgag tggtggtggt ggttccgacg tcgtgatgac ccaaag 46
<210> 45
<211> 49
<212> DNA
<213> Artificial Sequence
<220>
<223> PCR primer
<400> 45
cagtctcgag tggtggtggt ggtggttccg acgtcgtgat gacccaaag 49
<210> 46
<211> 52
<212> DNA
<213> Artificial Sequence
<220>
<223> PCR primer
<400> 46
cagtctcgag tggtggtggt ggtggtggtt ccgacgtcgt gatgacccaa ag
<210> 47
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> PCR primer
<400> 47
EP 1 327 681 A1
ggccgcatgt tgtcacgaat 20
<210> 48
<211> 780
<212> DNA
<213> Mus
<220>
<221> CDS
<222> (1). . . (768)
<223> CF2HL-0/pCOSl. MABL2-scFv<HL~0>
<400> 48
atg gga tgg age tgt ate ate etc ttc ttg gta gca aca get aca ggt gtc 51
MET Gly Trp Ser Cys He lie Leu Phe Leu Val Ala Thr Ala Thr Gly Val
5 10 15
gac tec cag gtc cag ctg cag cag tct gga cct gaa ctg gta aag cct ggg 102
Asp Ser Gin Val Gin Leu Gin Gin Ser Gly Pro Glu Leu Val Lys Pro Gly
20 25 30
get tea gtg aag atg tec tgc aag get tct gga tac acc ttc get aac cat 153
Ala Ser Val Lys MET Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ala Asn His
35 40 45 50
gtt att cac tgg gtg aag cag aag cca ggg cag ggc ctt gag tgg att gga 204
Val lie His Trp Val Lys Gin Lys Pro Gly Gin Gly Leu Glu Trp He Gly
55 60 65
tat att tat cct tac aat gat ggt act aag tat aat gag aag ttc aag gac 255
Tyr He Tyr Pro Tyr Asn Asp Gly Thr Lys Tyr Asn Glu Lys Phe Lys Asp
70 75 80 85
aag gec act ctg act tea gac aaa tec tec acc aca gec tac atg gac etc 306
Lys Ala Thr Leu Thr Ser Asp Lys Ser Ser Thr Thr Ala Tyr MET Asp Leu
72
EP 1 327 681 A1
90 95 100
age age ctg gec tct gag gac tct gcg gtc tat tac tgt gca aga ggg ggt 357
Ser Ser Leu Ala Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg Gly Gly
105 110 115
tac tat act tac gac gac tgg ggc caa ggc acc act etc aca gtc teg agt 408
Tyr Tyr Thr Tyr Asp Asp Trp Gly Gin Gly Thr Thr Leu Thr Val Ser Ser
120 125 130 135
gac gtc gtg atg acc caa agt cca etc tec ctg cct gtc agt ctt gga gat 459
Asp Val Val MET Thr Gin Ser Pro Leu Ser Leu Pro Val Ser Leu Gly Asp
140 145 150
caa gec tec ate tct tgc aga tea agt cag age ctt gtg cac agt aat gga 510
Gin Ala Ser lie Ser Cys Arg Ser Ser Gin Ser Leu Val His Ser Asn Gly
155 160 165 170
aag acc tat tta cat tgg tac ctg cag aag cca ggc cag tct cca aaa etc 561
Lys Thr Tyr Leu His Trp Tyr Leu Gin Lys Pro Gly Gin Ser Pro Lys Leu
175 180 185
ctg ate tac aaa gtt tec aac cga ttt tct ggg gtc cca gac agg ttc agt 612
Leu lie Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro Asp Arg Phe Ser
190 195 200
ggc agt gga tea gtg aca gat ttc aca etc atg ate age aga gtg gag get 663
Gly Ser Gly Ser Val Thr Asp Phe Thr Leu MET lie Ser Arg Val Glu Ala
205 210 215 220
gag gat ctg gga gtt tat ttc tgc tct caa agt aca cat gtt ccg tac acg 714
Glu Asp Leu Gly Val Tyr Phe Cys Ser Gin Ser Thr His Val Pro Tyr Thr
225 230 235
ttc gga ggg ggg acc aag ctg gaa ata aaa gac tac aaa gac gat gac gat 765
Phe Gly Gly Gly Thr Lys Leu Glu lie Lys Asp Tyr Lys Asp Asp Asp Asp
240 245 250 255
73
EP 1 327 681 A1
aaa taa tga gga tec 780
Lys
<210> 49
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> PCR primer
<400> 49
caagctcgag ataaaatccg gaggecaggt ecaattgeag cagtc 45
<210> 50
<211> 48
<212> DNA
<213> Artificial Sequence
<220>
<223> PCR primer
<400> 50
caagctcgag ataaaatccg gaggtggcca ggtccaattg cagcagtc
<210> 51
<211> 51
<212> DNA
<213> Artificial Sequence
<220>
<223> PCR primer
<400> 51
74
EP 1 327 681 A1
caagctcgag ataaaatccg gaggtggtgg ccaggtccaa ttgcagcagt c 51
<210> 52
<211> 54
<212> DNA
<213> Artificial Sequence
<220>
<223> PCR primer
<400> 52
caagctcgag ataaaatccg gaggtggtgg tggccaggtc caattgcagc agtc 54
<210> 53
<211> 57
<212> DNA
<213> Artificial Sequence
<220>
<223> PCR primer
<400> 53
caagctcgag ataaaatccg gaggtggtgg tggtggccag gtccaattgc agcagtc
<210> 54
<211> 780
<212> DNA
<213> Mus
<220>
<221> CDS
<222> (1)... (768)
<223> CF2LH-0/pCOSl. MABL2-scFv<LH-0>
75
EP 1 327 681 A1
<400> 54
atg aag ttg cct gtt agg ctg ttg gtg ctg atg ttc tgg att cct ggt tec 51
MET Lys Leu Pro Val Arg Leu Leu Val Leu MET Phe Trp He Pro Gly Ser
5 10 15
age agt gat gtt gtg atg acc caa agt cca etc tec ctg cct gtc agt ctt 102
Ser Ser Asp Val Val MET Thr Gin Ser Pro Leu Ser Leu Pro Val Ser Leu
20 25 30
gga gat caa gee tec ate tct tgc aga tea agt cag age ctt gtg cac agt 153
Gly Asp Gin Ala Ser He Ser Cys Arg Ser Ser Gin Ser Leu Val His Ser
35 40 45 50
aat gga aag acc tat tta cat tgg tac ctg cag aag cca ggc cag tct cca 204
Asn Gly Lys Thr Tyr Leu His Trp Tyr Leu Gin Lys Pro Gly Gin Ser Pro
55 60 65
aaa etc ctg ate tac aaa gtt tec aac cga ttt tct ggg gtc cca gac agg 255
Lys Leu Leu lie Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro Asp Arg
70 75 80 85
ttc agt ggc agt gga tea gtg aca gat ttc aca etc atg ate age aga gtg 306
Phe Ser Gly Ser Gly Ser Val Thr Asp Phe Thr Leu MET He Ser Arg Val
90 95 100
gag get gag gat ctg gga gtt tat ttc tgc tct caa agt aca cat gtt ccg 357
Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser Gin Ser Thr His Val Pro
105 110 115
tac acg ttc gga ggg ggg acc aag etc gag ata aaa cag gtc caa ttg cag 408
Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu He Lys Gin Val Gin Leu Gin
120 125 130 135
cag tct gga cct gaa ctg gta aag cct ggg get tea gtg aag atg tec tgc 459
Gin Ser Gly Pro Glu Leu Val Lys Pro Gly Ala Ser Val Lys MET Ser Cys
140 145 150
76
EP 1 327 681 A1
aag get tct gga tac acc ttc get aac cat gtt att cac tgg gtg aag cag 510
Lys Ala Ser Gly Tyr Thr Phe Ala Asn His Val lie His Trp Val Lys Gin
155 160 165 170
aag cca ggg cag ggc ctt gag tgg att gga tat att tat cct tac aat gat 561
Lys Pro Gly Gin Gly Leu Glu Trp lie Gly Tyr lie Tyr Pro Tyr Asn Asp
175 180 185
ggt act aag tat aat gag aag ttc aag gac aag gec act ctg act tea gac 612
Gly Thr Lys Tyr Asn Glu Lys Phe Lys Asp Lys Ala Thr Leu Thr Ser Asp
190 195 200
aaa tec tec acc aca gec tac atg gac etc age age ctg gee tct gag gac 663
Lys Ser Ser Thr Thr Ala Tyr MET Asp Leu Ser Ser Leu Ala Ser Glu Asp
205 210 215 220
tct gcg gtc tat tac tgt gca aga ggg ggt tac tat act tac gac gac tgg 714
Ser Ala Val Tyr Tyr Cys Ala Arg Gly Gly Tyr Tyr Thr Tyr Asp Asp Trp
225 230 235
ggc caa ggc acc act etc aca gtc tec tea gac tac aaa gac gat gac gat 765
Gly Gin Gly Thr Thr Leu Thr Val Ser Ser Asp Tyr Lys Asp Asp Asp Asp
240 245 250 255
aaa taa tga gga tec 780
Lys
<210> 55
<211> 351
<212> DNA
<213> Human
<220>
<221> CDS
<222> (1). . . (351)
77
EP 1 327 681 A1
10
15
<223> 12B5HV. 1-351 peptide
<400> 55
cag gtg cag ctg gtg cag tct ggg gga ggc ttg gtc egg ccc ggg ggg 48
Gin Val Gin Leu Val Gin Ser Gly Gly Gly Leu Val Arg Pro Gly Gly
15 10 15
tec ctg agt etc tec tgt gca gtc tct gga ate acc etc agg acc tac 96
Ser Leu Ser Leu Ser Cys Ala Val Ser Gly He Thr Leu Arg Thr Tyr
20 25 30
ggc atg cac tgg gtc cgc cag get cca ggc aag ggg ctg gag tgg gtg 144
20 Gly Met His Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
gca ggt ata tec ttt gac gga aga agt gaa tac tat gca gac tec gtg 192
25
Ala Gly lie Ser Phe Asp Gly Arg Ser Glu Tyr Tyr Ala Asp Ser Val
50 55 60
cag ggc cga ttc acc ate tec aga gac agt tec aag aac acc ctg tat 240
30
Gin Gly Arg Phe Thr He Ser Arg Asp Ser Ser Lys Asn Thr Leu Tyr
65 70 75 80
35 ctg caa atg aac age ctg aga gee gag gac acg get gtg tat tac tgt 288
Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
gcg aga gga gca cat tat ggt ttc gat ate tgg ggc caa ggg aca atg 336
Ala Arg Gly Ala His Tyr Gly Phe Asp He Trp Gly Gin Gly Thr Met
100 105 110
gtc acc gtc teg agt 351
Val Thr Val Ser Ser
50 115
40
45
<210> 56
55
78
EP 1 327 681 A1
<211> 57
<212> DNA
<213> Human
<220>
<221> CDS
<222> (1). . . (57)
<223> reader sequence
<400> 56
atg gag ttt ggg ctg age tgg gtt ttc etc gtt get ctt tta aga ggt 48
Met Glu Phe Gly Leu Ser Trp Val Phe Leu Val Ala Leu Leu Arg Gly
5 10 15
gtc cag tgt 57
Val Gin Cys
<210> 57
<211> 115
<212> DNA
<213> Artificial Sequence
<220>
<223> 12B5VH-1
<400> 57
atggagtttg ggctgagctg ggttttcctc gttgctcttt taagaggtgt ccagtgtcag 60
gtgcagctgg tgcagtctgg gggaggcttg gtccggcccg gggggtccct gagtc 115
<210> 58
<211> 115
<212> DNA
79
EP 1 327 681 A1
<213> Artificial Sequence
<220>
<223> 12B5VH-2
<400> 58
aaggatatac ctgccaccca ctccagcccc ttgcctggag cctggcggac ccagtgcatg 60
ccgtaggtcc tgagggtgat tccagagact gcacaggaga gactcaggga ccccc 115
<210> 59
<211> 115
<212> DNA
<213> Artificial Sequence
<220>
<223> 12B5VH-3
<400> 59
ggcaggtata tcctttgacg gaagaagtga atactatgca gactccgtgc agggccgatt 60
caccatctcc agagacagtt ccaagaacac cctgtatctg caaatgaaca gcctg 115
<210> 60
<211> 108
<212> DNA
<213> Artificial Sequence
<220>
<223> 12B5VH-4
<400> 60
actcgagacg gtgaccattg tcccttggcc ccagatatcg aaaccataat gtgctcctct 60
cgcacagtaa tacacagccg tgtcctcggc tctcaggctg ttcatttg 108
<210> 61
80
EP 1 327 681 A1
<211> 32
5 <212> DNA
<213> Artificial Sequence
<220>
10 <223> 12B5VH-S, PCR primer
<400> 61
ttcaagcttc caccatggag tttgggctga gc 32
15
<210> 62
20 <211> 34
<212> DNA
<213> Artificial Sequence
25 <220>
<223> 12B5VH-A, PCR primer
<400> 62
30
ttgggatcca ctcaccactc gagacggtga ccat 34
<210> 63
<211> 588
<212> DNA
<213> Human
<220>
<221> CDS
<222> (236) . . . (558)
<223> 1-235; intron, 236-558; Human IgG constant region (partial)
<400> 63
gaattcgtga gtggatccca agctagcttt ctggggcagg ccaggcctga ccttggcttt 60
ggggcaggga gggggctaag gtgaggcagg tggcgccagc caggtgcaca cccaatgccc 120
81
EP 1 327 681 A1
atgagcccag acactggacg ctgaacctcg cggacagtta agaacccagg ggcctctgcg
ccctgggccc agctctgtcc cacaccgcgg tcacatggca caacctctct tgca gcc
Ala
1
tec acc aag ggc cca teg gtc ttc ccc ctg gca ccc tec tec aag age
Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser
5 10 15
acc tct ggg ggc aca gcg gcc ctg ggc tgc ctg gtc aag gac tac ttc
Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe
20 25 30
ccc gaa ccg gtg acg gtg teg tgg aac tea ggc gcc ctg acc age ggc
Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly
35 40 45
gtg cac acc ttc ccg get gtc eta cag tec tea gga etc tac tec etc
Val His Thr Phe Pro Ala Val Leu Gin Ser Ser Gly Leu Tyr Ser Leu
50 55 60 65
age age gtg gtg acc gtg ccc tec age age ttg ggc acc cag acc tac
Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gin Thr Tyr
70 75 80
ate tgc aac gtg aat cac aag ccc age aac acc aag gtg gac aag aaa
lie Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys
85 90 95
gtt gag ccc aaa tct tgt gac aaa act cac aca
Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr
100 105
<210> 64
<211> 27
82
EP 1 327 681 A1
<212> DNA
<213> Artificial Sequence
<220>
<223> G1CH1-S, PCR primer
<400> 64
tgagaattcg tgagtggatc ccaagct 27
<210> 65
<211> 60
<£12> DNA
<213> Artificial Sequence
<220>
<223> G1CH1-A, PCR primer
<400> 65
aaaagatctt tatcatgtgt gagttttgtc acaagatttg ggctcaactt tcttgtccac 60
<210> 66
<211> 432
<212> DNA
<213> Human
<220>
<221> CDS
<222> (12). . . (419)
<223> HEF-12B5H-g gamma. 12-419 peptide
<400> 66
aagcttccac c atg gag ttt ggg ctg age tgg gtt ttc etc gtt get ctt 50
Met Glu Phe Gly Leu Ser Trp Val Phe Leu Val Ala Leu
1 5 10
83
EP 1 327 681 A1
tta aga ggt gtc cag tgt cag gtg cag ctg gtg cag tct ggg gga ggc 98
Leu Arg Gly Val Gin Cys Gin Val Gin Leu Val Gin Ser Gly Gly Gly
15 20 25
ttg gtc egg ccc ggg ggg tec ctg agt etc tec tgt gca gtc tct gga 146
Leu Val Arg Pro Gly Gly Ser Leu Ser Leu Ser Cys Ala Val Ser Gly
30 35 40 45
ate ace etc agg ace tac ggc atg cac tgg gtc cgc cag get cca ggc 194
lie Thr Leu Arg Thr Tyr Gly Met His Trp Val Arg Gin Ala Pro Gly
50 55 . 60
aag ggg ctg gag tgg gtg gca ggt ata tec ttt gac gga aga agt gaa 242
Lys Gly Leu Glu Trp Val Ala Gly He Ser Phe Asp Gly Arg Ser Glu
65 70 75
tac tat gca gac tec gtg cag ggc cga ttc acc ate tec aga gac agt 290
Tyr Tyr Ala Asp Ser Val Gin Gly Arg Phe Thr He Ser Arg Asp Ser
80 85 90
tec aag aac acc ctg tat ctg caa atg aac age ctg aga gee gag gac 338
Ser Lys Asn Thr Leu Tyr Leu Gin Met Asn Ser Leu Arg Ala Glu Asp
95 100 105
acg get gtg tat tac tgt gcg aga gga gca cat tat ggt ttc gat ate 386
Thr Ala Val Tyr Tyr Cys Ala Arg Gly Ala His Tyr Gly Phe Asp He
110 115 120 125
tgg ggc caa ggg aca atg gtc acc gtc teg agt ggtgagtgga tec 432
Trp Gly Gin Gly Thr Met Val Thr Val Ser Ser
130 135
<210> 67
<211> 321
<212> DNA
84
EP 1 327 681 A1
10
15
<213> Human
<220>
<221> CDS
<222> (1).. . (321)
<223> 12B5LV. 1-321 peptide
<400> 67
gac ate cag atg acc cag tct cct tec acc ctg tct gca tct att gga 48
Asp He Gin Met Thr Gin Ser Pro Ser Thr Leu Ser Ala Ser lie Gly
1 5 10 15
20 gac aga gtc acc ate acc tgc egg gee age gag ggt att tat cac tgg 96
Asp Arg Val Thr lie Thr Cys Arg Ala Ser Glu Gly lie Tyr His Trp
20 25 30
ttg gee tgg tat cag cag aag cca ggg aaa gee cct aaa etc ctg ate 144
Leu Ala Trp Tyr Gin Gin Lys Pro Gly Lys Ala Pro Lys Leu Leu He
35 40 45
30
tat aag gec tct agt tta gee agt ggg gee cca tea agg ttc age ggc .192
Tyr Lys Ala Ser Ser Leu Ala Ser Gly Ala Pro Ser Arg Phe Ser Gly
50 55 60
agt gga tct ggg aca gat ttc act etc acc ate age age ctg cag cct 240
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr He Ser Ser Leu Gin Pro
65 70 75 80
gat gat ttt gca act tat tac tgc caa caa tat agt aat tat ccg etc 288
Asp Asp Phe Ala Thr Tyr Tyr Cys Gin Gin Tyr Ser Asn Tyr Pro Leu
85 90 95
act ttc ggc gga ggg acc aag ctg gag ate aaa 321
Thr Phe Gly Gly Gly Thr Lys Leu Glu He Lys
100 105
35
40
45
50
55
85
EP 1 327 681 A1
<210> 68
<2ll> 66
<212> DNA
<213> Human
<220>
<221> CDS
<222> (1)... (66)
<223> reader sequence
<400> 68
atg gac atg agg gtc ccc get cag etc ctg ggg etc ctg ctg etc tgg 48
MET Asp MET Arg Val Pro Ala Gin Leu Leu Gly Leu Leu Leu Leu Trp
5 10 15
etc cca ggt gec aaa tgt 66
Leu Pro Gly Ala Lys Cys
20
<210> 69
<211> 110
<212> DNA
<213> Artificial Sequence
<220>
<223> 12B5VL-1
<400> 69
atggacatga gggtccccgc tcagctcctg gggctcctgc tgctctggct cccaggtgcc I
aaatgtgaca tccagatgac ccagtctcct tccaccctgt ctgeatctat 1.
<210> 70
<211> 110
86
EP 1 327 681 A1
10
15
35
45
50
<212> DNA
<213> Artificial Sequence
<220>
<223> 12B5VL-2
<400> 70
ggagtttagg ggctttccct ggcttctgct gataccaggc caaccagtga taaataccct 60
cgctggcccg gcaggtgatg gtgactctgt ctccaataga tgcagacagg 110
<210> 71
20 <211> 110
<212> DNA
<213> Artificial Sequence
<220>
<223> 12B5VL-3
<400> 71
aagcccctaa actcctgatc tataaggcct ctagtttagc cagtggggcc ccatcaaggt 60
tcagcggcag tggatctggg acagatttca ctctcaccat cagcagcctg 110
<210> 72
<211> 103
40 <212> DNA
<213> Artificial Sequence
<220>
<223> 12B5VL-4
<400> 72
tttgatctcc agcttggtcc ctccgccgaa agtgagcgga taattactat attgttggca 60
gtaataagtt gcaaaatcat caggctgcag gctgctgatg gtg 103
87
EP 1 327 681 A1
<210> 73
s <211> 32
<212> DNA
<213> Artificial Sequence
10 <220>
<223> 12B5VL-S, PCR primer
<400> 73
15
ttcaagcttc caccatggac atgagggtcc cc 32
2 o <210> 74
<211> 35
<212> DNA
25 <213> Artificial Sequence
<220>
<223> 12B5VL-A, PCR primer
30
<400> 74
tctaggatcc actcacgttt gatctccagc ttggt 35
35
<210> 75
<211> 415
40 <212> DNA
<213> Human
<220>
45
<221> CDS
<222> (12)... (398)
<223> HEF-12B5H-g kappa. 12-398 peptide
<400> 75
aagcttccac c atg gac atg agg gtc ccc get cag etc ctg ggg etc ctg 50
88
EP 1 327 681 A1
Met Asp Met Arg Val Pro Ala Gin Leu Leu Gly Leu Leu
1 5 10
ctg etc tgg etc cca ggt gec aaa tgt gac ate cag atg ace cag tct 98
Leu Leu Trp Leu Pro Gly Ala Lys Cys Asp lie Gin Met Thr Gin Ser
15 20 25
cct tec acc ctg tct gca tct att gga gac aga gtc acc ate acc tgc 146
Pro Ser Thr Leu Ser Ala Ser He Gly Asp Arg Val Thr He Thr Cys
30 35 40 45
egg gec age gag ggt att tat cac tgg ttg gee tgg tat cag cag aag 194
Arg Ala Ser Glu Gly lie Tyr His Trp Leu Ala Trp Tyr Gin Gin Lys
50 55 60
cca ggg aaa gec cct aaa etc ctg ate tat aag gec tct agt tta gec 242
Pro Gly Lys Ala Pro Lys Leu Leu lie Tyr Lys Ala Ser Ser Leu Ala
65 70 75
agt ggg gee cca tea agg ttc age ggc agt gga tct ggg aca gat ttc 290
Ser Gly Ala Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe
80 85 90
act etc acc ate age age ctg cag cct gat gat ttt gca act tat tac 338
Thr Leu Thr He Ser Ser Leu Gin Pro Asp Asp Phe Ala Thr Tyr Tyr
95 100 105
tgc caa caa tat agt aat tat ccg etc act ttc ggc gga ggg acc aag 386
Cys Gin Gin Tyr Ser Asn Tyr Pro Leu Thr Phe Gly Gly Gly Thr Lys
110 115 120 125
ctg gag ate aaa cgtgagtgga tcctaga 415
Leu Glu He Lys
<210> 76
<211> 24
89
EP 1 327 681 A1
<212> DNA
<213> Artificial Sequence
<220>
<223> FLAG tag sequence
<400> 75
gac tac aag gat gac gac gat aag 24
Asp Tyr Lys Asp Asp Asp Asp Lys
5
<510> 77
<211> 31
<212> DNA
<213> Artificial Sequence
<220>
<223> 12B5-S, PCR primer
<400> 77
atagaattcc accatggagt ttgggctgag c 31
<210> 78
<211> 24
<212> DNA
<213> Artificial Sequence
<220>
<223> HuVHJ3, PCR primer
<400> 78
tgaagagacg gtgaccattg tccc 24
<210> 79
90
EP 1 327 681 A1
<211> 28
<212> DNA
<213> Artificial Sequence
<220>
<223> RhuJH3, PCR primer
<400> 79
ggacaatggt caccgtctct tcaggtgg 28
<210> 80
<211> 32
<212> DNA
<213> Artificial Sequence
<220>
<223> RhuVKl, PCR primer
<400> 80
ggagactggg tcatctggat gtccgatccg cc 32
<210> 81
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> HuVKl. 2, PCR primer
<400> 81
gacatccaga tgacccagtc tec 23
<210> 82
<211> 59
EP 1 327 681 A1
<212> DNA
5 <213> Artificial Sequence
<220>
<223> 12B5F-A, PCR primer
10 <400> 82
attgcggccg cttatcactt atcgtcgtca tccttgtagt ctttgatctc cagcttggt 59
15
<210> 83
<211> 45
20 <212> DNA
<213> Artificial Sequence
<220>
25
<223> Linker amino acid sequence and nucleotide sequence
<400> 83
ggt ggt ggt ggt teg ggt ggt ggt ggt teg ggt ggt ggc gga teg 45
30
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
5 10 15
35
<210> 84
<211> 823
40 <212> DNA
<213> Human
<220>
45
<221> CDS
<222> (12). .. (809)
50 <223> scl2B5, Single chain Fv
<400> 84
aagcttccac c atg gag ttt ggg ctg age tgg gtt ttc etc gtt get ctt 50
92
EP 1 327 681 A1
Met Glu Phe Gly Leu Ser Trp Val Phe Leu Val Ala Leu
1 5 10
tta aga ggt gtc cag tgt cag gtg cag ctg gtg cag tct ggg gga ggc 98
Leu Arg Gly Val Gin Cys Gin Val Gin Leu Val Gin Ser Gly Gly Gly
15 20 25
ttg gtc egg ccc ggg ggg tec ctg agt etc tec tgt gca gtc tct gga 146
Leu Val Arg Pro Gly Gly Ser Leu Ser Leu Ser Cys Ala Val Ser Gly
30 35 40 45
ate acc etc agg acc tac ggc atg cac tgg gtc cgc cag get cca ggc 194
He Thr Leu Arg Thr Tyr Gly Met His Trp Val Arg Gin Ala Pro Gly
50 " 55 60
aag ggg ctg gag tgg gtg gca ggt ata tec ttt gac gga aga agt gaa 242
Lys Gly Leu Glu Trp Val Ala Gly He Ser Phe Asp Gly Arg Ser Glu
65 70 75
tac tat gca gac tec gtg cag ggc cga ttc acc ate tec aga gac agt 290
Tyr Tyr Ala Asp Ser Val Gin Gly Arg Phe Thr He Ser Arg Asp Ser
80 85 90
tec aag aac acc ctg tat ctg caa atg aac age ctg aga gec gag gac 338
Ser Lys Asn Thr Leu Tyr Leu Gin Met Asn Ser Leu Arg Ala Glu Asp
95 100 105
acg get gtg tat tac tgt gcg aga gga gca cat tat ggt ttc gat ate 386
Thr Ala Val Tyr Tyr Cys Ala Arg Gly Ala His Tyr Gly Phe Asp He
110 115 120 125
tgg ggc caa ggg aca atg gtc acc gtc teg agt ggt ggt ggt ggt teg 434
Trp Gly Gin Gly Thr Met Val Thr Val Ser Ser Gly Gly Gly Gly Ser
130 135 140
ggt ggt ggt ggt teg ggt ggt ggc gga teg gac ate cag atg acc cag 482
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp He Gin Met Thr Gin
93
EP 1 327 681 A1
. 145 150 155
tct cct tec acc ctg tct gca tct att gga gac aga gtc acc ate acc 530
Ser Pro Ser Thr Leu Ser Ala Ser He Gly Asp Arg Val Thr lie Thr
160 165 170
tgc egg gec age gag ggt att tat cac tgg ttg gec tgg tat cag cag 578
Cys Arg Ala Ser Glu Gly He Tyr His Trp Leu Ala Trp Tyr Gin Gin
175 180 185
aag cca ggg aaa gec cct aaa etc ctg ate tat aag gee tct agt tta 626
Lys Pro Gly Lys Ala Pro Lys Leu Leu lie Tyr Lys. Ala Ser Ser Leu
190 195 200 205
gec agt ggg gee cca tea "agg ttc age ggc agt gga tct ggg aca gat 674
Ala Ser Gly Ala Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp
210 215 220
ttc act etc acc ate age age ctg cag cct gat gat ttt gca act tat 722
Phe Thr Leu Thr He Ser Ser Leu Gin Pro Asp Asp Phe Ala Thr Tyr
225 230 235
TAC TGC CAA CAA TAT AGT AAT TAT CCG CTC ACT TTC GGC GGA GGG ACC 770
Tyr Cys Gin Gin Tyr Ser Asn Tyr Pro Leu Thr Phe Gly Gly Gly Thr
240 245 250
aag ctg gag ate aaa gac tac aag gat gac gac gat aag tgataagegg c 820
Lys Leu Glu lie Lys Asp Tyr Lys Asp Asp Asp Asp Lys
255 260 265
cgc 823
<210> 85
<211> 114
<212> PRT
<213> Human
94
EP 1 327 681 A1
w
15
20
45
50
<400> 85
Gin Val Gin Leu Gin Gin Ser Gly Pro Gly Leu Val Lys Pro Ser Glu
15 10 15
Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Asp Ser lie Ser Ser Tyr
20 25 30
Tyr Trp Ser Trp He Arg Gin Pro Pro Gly Lys Gly Leu Glu Trp He
35 40 45
Gly Tyr He Tyr Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys
„ 50 55 60
Ser Arg Val Thr He Ser Val Asp Thr Ser Lys Ser Gin Phe Ser Leu
65 70 75 80
Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Arg Gly Arg Tyr Phe Asp Val Trp Gly Arg Gly Thr Met Val Thr Val
100 105 110
Ser Ser
35
<210> 86
<211> 342
40 <212> DNA
<213> Human
<400> 86
caggtgcagc tgcagcagtc gggcccagga ctggtgaagc cttcggagac cctgtccctc 60
acctgcactg tctctggtga ctccatcagt agttactact ggagctggat tcggcagccc 120
ccagggaagg gactggagtg gattgggtat atctattaca gtgggagcac caactacaac 180
ccctccctca agagtcgagt caccatatca gtagacacgt ccaagagcca gttctccctg 240
95
EP 1 327 681 A1
aagctgagct ctgtgaccgc cgcagacacg gccgtgtatt actgtgcgag agggcggtac 300
ttcgatgtct ggggccgtgg caccatggtc actgtctcct ca 342
<210> 87
<211> 57
<212> DNA
<213> Human
<220>
<221> CDS
<222> (1) . . . (57)
<223> reader sequence
<308> GenBank No. AF062252
<400> 87
atg aaa cat ctg tgg ttc ttc ctt etc ctg gtg gca get ccc aga tgg 48
Met Lys His Leu Trp Phe Phe Leu Leu Leu Val Ala Ala Pro Arg Trp
1 5 10 15
gtc ctg tec 57
Val Leu Ser
<210> 88
<211> 110
<212> DNA
<213> Artificial Sequence
<220>
<223> 12E10VH1
<400> 88
atgaaacatc tgtggttctt ccttctcctg gtggcagctc ccagatgggt cctgtcccag 60
gtgcagctgc ageagteggg cccaggactg gtgaagcett cggagaccct 110
96
EP 1 327 681 A1
<210> 89
<211> 110
<212> DNA
<213> Artificial Sequence
<220>
<223> 12E10VH2
<400> 89
acccaatcca ctccagtccc ttccctgggg gctgccgaat ccagctccag tagtaactac
tgatggagtc accagagaca gtgcaggtga gggacagggt ctccgaaggc
<210> 90
<211> 110
<212> DNA
<213> Artificial Sequence
<220>
<223> 12E10VH3
<400> 90
tggagtggat tgggtatatc tattacagtg ggagcaccaa ctacaacccc tccctcaaga
gtcgagtcac catatcagta gacacgtcca agagccagtt ctccctgaag
<210> 91
<211> 114
<212> DNA
<213> Artificial Sequence
<220>
<223> 12E10VH4
<400> 91
97
EP 1 327 681 A1
tgaggagaca gtgaccatgg tgccacggcc ccagacatcg aagtaccgcc ctctcgcaca 60
gtaatacacg gccgtgtctg cggcggtcac agagctcagc ttcagggaga actg 114
<210> 92
<211> 32
<212> DNA
<213> Artificial Sequence
<220>
<223> 12E10VHS, PCR primer
<400> 92
ttcaagcttc caccatgaaa catctgtggt tc 32
<210> 93
<211> 34
<212> DNA
30
<213> Artificial Sequence
<220>
35 <223> 12E10VHA, PCR primer
<400> 93
ttgggatcca ctcacctgag gagacagtga ccat 34
40
<210> 94
<211> 426
45
<212> DNA
<213> Mus
<220>
<221> CDS
<222> (12). . . (417)
98
EP 1 327 681 A1
<223> 12E10H, H chain V region
<400> 94
aagcttccac c atg aaa cat ctg tgg ttc ttc ctt etc ctg gtg gca get 50
Met Lys His Leu Trp Phe Phe Leu Leu Leu Val Ala Ala
1 5 10
ccc aga tgg gtc ctg tec cag gtg cag ctg cag cag teg ggc cca gga 98
Pro Arg Trp Val Leu Ser Gin Val Gin Leu Gin Gin Ser Gly Pro Gly
15 20 25
ctg gtg aag cct teg gag ace ctg tec etc ace tgc act gtc tct ggt 146
Leu Val Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys Thr Val Ser Gly
30 35 40 45
gac tec ate agt agt tac tac tgg age tgg att egg cag ccc cca ggg 194
Asp Ser He Ser Ser Tyr Tyr Trp Ser Trp lie Arg Gin Pro Pro Gly
50 55 60
aag gga ctg gag tgg att ggg tat ate tat tac agt ggg age acc aac 242
Lys Gly Leu Glu Trp lie Gly Tyr He Tyr Tyr Ser Gly Ser Thr Asn
65 70 75
tac aac ccc tec etc aag agt cga gtc acc ata tea gta gac acg tec 290
Tyr Asn Pro Ser Leu Lys Ser Arg Val Thr He Ser Val Asp Thr Ser
80 85 90
aag age cag ttc tec ctg aag ctg age tct gtg acc gee gca gac acg 338
Lys Ser Gin Phe Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr
95 100 105
gec gtg tat tac tgt gcg aga ggg egg tac ttc gat gtc tgg ggc cgt 386
Ala Val Tyr Tyr Cys Ala Arg Gly Arg Tyr Phe Asp Val Trp Gly Arg
110 115 120 125
ggc acc atg gtc act gtc tec tea ggtgagtgga tcccaa 426
Gly Thr Met Val Thr Val Ser Ser
99
EP 1 327 681 A1
130
<210> 95
<211> 110
<212> PRT
<213> Mus
<400> 95
Ser Tyr Val Leu Thr Gin Pro Pro Ser Val Ser Gly Ser Pro Gly Gin
.1 5 10 15
Ser He Thr He Ser Cys'Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr
20 25 30
Asn Tyr Val Ser Trp Tyr Gin Gin His Pro Gly Lys Ala Pro Lys Leu
35 40 45
Met He Tyr Glu Gly Ser Lys Arg Pro Ser Gly Val Ser Asn Arg Phe
50 55 60
Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr He Ser Gly Leu
65 70 75 80
Gin Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Thr Thr Arg
85 90 95
Ser Thr Arg Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu
100 105 110
<210> 96
<211> 330
<212> DNA
<213> Mus
100
EP 1 327 681 A1
<400> 96
tcctatgtgc
tgactcagcc
accctcggtg
tcagggtctc
ctggacagtc
gatcaccatc
60
tcctgcactg
gaaccagcag
tgacgttggt
ggttataact
atgtctcctg
gtaccaacag
120
cacccaggca
aagcccccaa
actcatgatt
tatgagggca
gtaaacggcc
ctcaggggtt
180
tctaatcgct
tctctggctc
caagtctggc
aacacggcct
ccctgaccat
ctctgggctc
240
caggctgagg
acgaggctga
ttattactgc
agctcatata
caaccagaag
cactcgggtg
300
ttcggcggag
ggaccaagct
gaccgtccta
330
<210> 97
<211> 57
<212> DNA
<213> Human
<220>
<221> CDS
<222> (1). . . (57)
<223> reader sequence
<310>
<400> 97
atg gcc tgg acc gtt etc etc etc ggc etc etc tct cac tgc aca ggc 48
Met Ala Trp Thr Val Leu Leu Leu Gly Leu Leu Ser His Cys Thr Gly
15 10 15
tct gtg acc 57
Ser Val Thr
<210> 98
<211> 110
<212> DNA
<213> Artificial Sequence
101
EP 1 327 681 A1
<220>
<223> 12E10VL1, PCR primer
<400> 98
atggcctgga ccgttctcct cctcggcctc ctctctcact gcacaggctc tgtgacctcc
tatgtgctga ctcagccacc ctcggtgtca gggtctcctg gacagtcgat
<210> 99
<211> 62
<212> DNA
<213> Artificial Sequence
<220>
<223> 12E10VL2, PCR primer
<400> 99
tcatgagttt gggggctttg cctgggtgct gttggtacca ggagacatag ttataaccac
caacgtcact gctggttcca gtgcaggaga tggtgatcga ctgtccagga
<210> 100
<211> 110
<212> DNA
<213> Artificial Sequence
<220>
<223> 12E10VL3, PCR primer
<400> 100
cccccaaact catgatttat gagggcagta aacggccctc aggggtttct aatcgcttct
ctggctccaa gtctggcaac acggcctccc tgaccatctc tgggctccag
<210> 101
<211> 102
102
EP 1 327 681 A1
<212> DNA
<213> Artificial Sequence
<220>
<223> 12E10VL4, PCR primer
<400> 101
taggacggtc agcttggtcc ctccgccgaa cacccgagtg cttctggttg tatatgagct
gcagtaataa tcagcctcgt cctcagcctg gagcccagag at
<210> 102
<211> 31
<212> DNA
<213> Artificial Sequence
<220>
<223> 12E10VLS, PCR primer
<400> 102
atcaagcttc caccatggcc tggaccgttc t 31
<210> 103
<211> 36
<212> DNA
<213> Artificial Sequence
<220>
<223> 12E10VLA, PCR primer
<400> 103
ctaggatccg ggctgaccta ggacggtcag cttggt 36
<210> 104
<211> 387
103
EP 1 327 681 A1
10
15
<212> DNA
<213> Mus
<220>
<221> CDS
<222> (1). . . (387)
<223> 12E10L, L chain V region
<310>
<400> 104
atg gcc tgg acc gtt etc etc etc ggc etc etc tct cac tgc aca ggc 48
20 Met Ala Trp Thr Val Leu Leu Leu Gly Leu Leu Ser His Cys Thr Gly
1 5 10 15
tct gtg acc tec tat gtg ctg act cag cca ccc teg gtg tea ggg tct 96
25
Ser Val Thr Ser Tyr Val Leu Thr Gin Pro Pro Ser Val Ser Gly Ser
20 25 30
3o cct gga cag teg ate .acc ate tec tgc act gga acc age agt gac gtt 144
Pro Gly Gin Ser He Thr He Ser Cys Thr Gly Thr Ser Ser Asp Val
35 40 45
35 ggt ggt tat aac tat gtc tec tgg tac caa cag cac cca ggc aaa gcc 192
Gly Gly Tyr Asn Tyr Val Ser Trp Tyr Gin Gin His Pro Gly Lys Ala
50 55 60
ccc aaa etc atg att tat gag ggc agt aaa egg ccc tea ggg gtt tct 240
Pro Lys Leu Met He Tyr Glu Gly Ser Lys Arg Pro Ser Gly Val Ser
65 70 75 80
aat cgc ttc tct ggc tec aag tct ggc aac acg gcc tec ctg acc ate 288
Asn Arg Phe Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr He
so 85 90 95
tct ggg etc cag get gag gac gag get gat tat tac tgc age tea tat 336
Ser Gly Leu Gin Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr
40
45
104
EP 1 327 681 A1
w
15
100 105 110
Aca acc aga age act egg gtg ttc ggc gga ggg ace aag ctg acc gtc 384
Thr Thr Arg Ser Thr Arg Val Phe Gly Gly Gly Thr Lys Leu Thr Val
115 120 125 .
eta 387
Leu
<210> 105
<211> 24
20 <212> DNA
<213> Artificial Sequence
<220>
<221> CDS
<222> (1)... (24)
<223> FLAG, reader sequence
30
<400> 105
gac tac aag gat gac gac gat aag 24
35 Asp Tyr Lys Asp Asp Asp Asp Lys
<210> 106
<211> 30
<212> DNA
<213> Artificial Sequence
<220>
<223> 12E10S, PCR primer
<400> 106
tatgaattcc accatgaaac atctgtggtt 30
105
EP 1 327 681 A1
<210> 107
<211> 38
<212> DNA
<213> Artificial Sequence
<220>
<223> DB2, PCR primer
<400> 107
taggagctac cgcctccacc tgaggagaca gtgaccat 38
<-210> 108
<211> 44
<212> DNA
<213> Artificial Sequence
<220>
<223> DB1, PCR primer
<400> 108
gtctcctcag gtggaggcgg tagctcctat gtgctgactc agcc 44
<210> 109
<211> 59
<212> DNA
<213> Artificial Sequence
<220>
<223> 12E10FA, PCR primer
<400> 109
attgcggccg cttatcactt atcgtcgtca tccttgtagt ctaggacggt cagcttggt
<210> 110
106
EP 1 327 681 A1
10
15
<211> 792
<212> DNA
<213> Artificial Sequence
<220>
<221> CDS
<222> (11). . . (778)
<223> 12E10, Single chain Fv
<400> 110
gaattccacc atg aaa cat ctg tgg ttc ttc ctt etc ctg gtg gca get 49
20 .. Met Lys His Leu Trp Phe Phe Leu Leu Leu Val Ala Ala
15 10
ccc aga tgg gtc ctg tec cag gtg cag ctg cag cag teg ggc cca gga 97
Pro Arg Trp Val Leu Ser Gin Val Gin Leu Gin Gin Ser Gly Pro Gly
15 20 25
ctg gtg aag cct teg gag acc ctg tec etc ace tgc act gtc tct ggt 145
30
Leu Val Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys Thr Val Ser Gly
30 35 40 45
35 gac tec ate agt agt tac tac tgg age tgg att egg cag ccc cca ggg 193
Asp Ser lie Ser Ser Tyr Tyr Trp Ser Trp He Arg Gin Pro Pro Gly
50 55 60
aag gga ctg gag tgg att ggg tat ate tat tac agt ggg age acc aac 241
Lys Gly Leu Glu Trp lie Gly Tyr He Tyr Tyr Ser Gly Ser Thr Asn
65 70 75
tac aac ccc tec etc aag agt cga gtc acc ata tea gta gac acg tec 289
Tyr Asn Pro Ser Leu Lys Ser Arg Val Thr He Ser Val Asp Thr Ser
80 85 90
aag age cag ttc tec ctg aag ctg age tct gtg acc gee gca gac acg 337
Lys Ser Gin Phe Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr
40
45
50
107
EP 1 327 681 A1
95 100 105
gcc gtg tat -tac tgt gcg aga ggg egg tac ttc gat gtc tgg ggc cgt 385
Ala Val Tyr Tyr Cys Ala Arg Gly Arg Tyr Phe Asp Val Trp Gly Arg
110 115 120 125
ggc acc atg gtc act gtc tec tea ggt gga ggc ggt age tec tat gtg 433
Gly Thr Met Val Thr Val Ser Ser Gly Gly Gly Gly Ser Ser Tyr Val
130 135 140
ctg act cag cca ccc teg gtg tea ggg tct cct gga cag teg ate acc 481
Leu Thr Gin Pro Pro Ser Val Ser Gly Ser Pro Gly Gin Ser He Thr
145 150 155
ate tec tgc act gga acc age agt gac gtt ggt ggt tat aac tat gtc 529
He Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr Asn Tyr Val
160 165 170
tec tgg tac caa cag cac cca ggc aaa gcc ccc aaa etc atg att tat 577
Ser Trp Tyr Gin Gin His Pro Gly Lys Ala Pro Lys Leu Met lie Tyr
175 180 185
gag ggc agt aaa egg ccc tea ggg gtt tct aat cgc ttc tct ggc tec 625
Glu Gly Ser Lys Arg Pro Ser Gly Val Ser Asn Arg Phe Ser Gly Ser
190 195 200 205
aag tct ggc aac acg gcc tec ctg acc ate tct ggg etc cag get gag 673
Lys Ser Gly Asn Thr Ala Ser Leu Thr lie Ser Gly Leu Gin Ala Glu
210 215 220
gac gag get gat tat tac tgc age tea tat aca acc aga age act egg 721
Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Thr Thr Arg Ser Thr Arg
225 230 235
gtg ttc ggc gga ggg acc aag ctg acc gtc eta gac tac aag gat gac 769
Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Asp Tyr Lys Asp Asp
240 245 250
108
EP 1 327 681 A1
gac gat aag tgataagcgg ccgc 792
Asp Asp Lys
255
<210> 111
<211> 62
<212> DNA
<213> Artificial Sequence
<220>
<223> sc4. 3, PCR primer
<400> 111
ggtggctgag tcagcacata ggacgatccg ccaccacccg aaccaccacc acccgaacca 60
cc 62
<210> 112
<211> 61
<212> DNA
<213> Artificial Sequence
<220>
<223> scl. 3, PCR primer
<400> 112
gcaccatggt cactgtctcc tcaggtggtg gtggttcggg tggtggtggt tcgggtggtg 60
g 61
<210> 113
<211> 822
<212> DNA
<213> Artificial Sequence
109
EP 1 327 681 A1
10
15
<220>
<221> CDS
<222> (11). . . (807)
<223> scl2E10, Single chain Fv
<400> 113
gaattccacc atg aaa cat ctg tgg ttc ttc ctt etc ctg gtg gca get 49
Met Lys His Leu Trp Phe Phe Leu Leu Leu Val Ala Ala
1 5 10
ccc aga tgg gtc ctg tec cag gtg cag ctg cag cag teg ggc cca gga 97
20 Pro Arg Trp Val Leu Ser Gin Val Gin Leu Gin Gin Ser Gly Pro Gly
15 ' 20 25
ctg gtg aag cct teg gag acc ctg tec etc acc tgc act gtc tct ggt 145
25
Leu Val Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys Thr Val Ser Gly
30 35 40 45
30 gac tec ate agt agt tac tac tgg age tgg att egg cag ccc cca ggg 193
Asp Ser lie Ser Ser Tyr Tyr Trp Ser Trp lie Arg Gin Pro Pro Gly
50 55 60
35 aag gga ctg gag tgg att ggg tat ate tat tac agt ggg age acc aac 241
Lys Gly Leu Glu Trp lie Gly Tyr He Tyr Tyr Ser Gly Ser Thr Asn
65 70 75
tac aac ccc tec etc aag agt cga gtc acc ata tea gta gac acg tec 289
Tyr Asn Pro Ser Leu Lys Ser Arg Val Thr He Ser Val Asp Thr Ser
80 85 90
aag age cag ttc tec ctg aag ctg age tct gtg acc gec gca gac acg 337
Lys Ser Gin Phe Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr
so 95 100 105
gec gtg tat tac tgt gcg aga ggg egg tac ttc gat gtc tgg ggc cgt 385
Ala Val Tyr Tyr Cys Ala Arg Gly Arg Tyr Phe Asp Val Trp Gly Arg
40
45
110
EP 1 327 681 A1
10
15
110 115 120 . 125
ggc acc atg gtc act gtc tec tea ggt ggt ggt ggt teg ggt ggt ggt 433
Gly Thr Met Val Thr Val Ser Ser Gly Gly Gly. Gly Ser Gly Gly Gly
130 135 140
ggt teg ggt ggt ggc gga teg tec tat gtg ctg act cag cca ccc teg 481
Gly Ser Gly Gly Gly Gly Ser Ser Tyr Val Leu Thr Gin Pro Pro Ser
145 150 155
gtg tea ggg tct cct gga cag teg ate acc ate tec tgc act gga acc 529
Val Ser Gly Ser Pro Gly Gin Ser lie Thr lie Ser Cys Thr Gly Thr
20 - 160 165 170
age agt gac gtt ggt ggt tat aac tat gtc tec tgg tac caa cag cac 577
Ser Ser Asp Val Gly Gly Tyr Asn Tyr Val Ser Trp Tyr Gin Gin His
175 180 185
cca ggc aaa gec ccc aaa etc atg att tat gag ggc agt aaa egg ccc 625
3o Pro Gly Lys Ala Pro Lys Leu Met He Tyr Glu Gly Ser Lys Arg Pro
190 195 200 205
tea ggg gtt tct aat cgc ttc tct ggc tec aag tct ggc aac acg gec 673
35 Ser Gly Val Ser Asn Arg Phe Ser Gly Ser Lys Ser Gly Asn Thr Ala
210 215 220
tec ctg acc ate tct ggg etc cag get gag gac gag get gat tat tac 721
Ser Leu Thr He Ser Gly Leu Gin Ala Glu Asp Glu Ala Asp Tyr Tyr
225 230 235
tgc age tea tat aca acc aga age act egg gtg ttc ggc gga ggg acc 769
Cys Ser Ser Tyr Thr Thr Arg Ser Thr Arg Val Phe Gly Gly Gly Thr
240 245 250
so aag ctg acc gtc eta gac tac aag gat gac gac gat aag tgataagegg 818
Lys Leu Thr Val Leu Asp Tyr Lys Asp Asp Asp Asp Lys
255 260 265
40
45
111
EP 1 327 681 A1
ccgc 822
Claims
1. A modified antibody comprising two or more H chain V regions and two or more L chain V regions of the same or
10 different monoclonal antibody and showing an agonist action by crosslinking a cell surface molecule(s) or intrac-
ellular molecule(s).
2. The modified antibody comprising two or more H chain V regions and two or more L chain V regions of monoclonal
antibody and showing an agonist action by crosslinking a cell surface molecule(s).
15
3. The modified antibody of claim 1 or 2, wherein the H chain V region and the L chain V region are connected through
a linker.
4. The modified antibody of claim 3, wherein the linker is a peptide linker comprising at least one amino acid.
20
5. The modified antibody of any one of claims 1 to 4, wherein the modified monoclonal antibody is a multimer of single
chain Fv comprising an H chain V region and an L chain V region.
6. The modified antibody of claim 5, wherein the modified antibody is composed of tetramer, trimer or dimer of single
25 chain Fv.
7. The modified antibody of claim 6, wherein the modified antibody is composed of dimer of single chain Fv.
8. The modified antibody of any one of claims 5 to 7, wherein the H chain V region and the L chain V region existing
30 in the same chain are not associated to form an antigen-binding site.
9. The modified antibody of any one of claims 1 to 4, wherein the modified antibody is a single chain polypeptide
comprising two or more H chain V regions and two or more L chain V regions.
35 10. The modified antibody of claim 9, wherein the modified antibody is a single chain polypeptide comprising two H
chain V regions and two L chain V regions.
11. The modified antibody of any one of claims 1 to 10, wherein the modified antibody further comprises an amino
acid sequence(s) for peptide purification.
40
12. The modified antibody of any one of claims 1 to 11 , wherein the modified antibody has been purified.
13. The modified antibody of any one of claims 1 to 12, wherein H chain V region and/or L chain V region is H chain
V region and/or L chain V region derived from a human antibody.
45
14. The modified antibody of any one of claims 1 to 13, wherein H chain V region and/or L chain V region is humanized
H chain V region and/or L chain V region.
15. The modified antibody of any one of claims 1 to 14, wherein the cell surface molecule or intracellular molecule is
so a hormone receptor, a cytokine receptor, tyrosine kinase receptor or intranuclear receptor.
16. The modified antibody of any one of claims 1 to 15, wherein the cell surface molecule or intracellular molecule is
erythropoietin (EPO) receptor, thrombopoietin (TPO) receptor, granulocyte colony stimulating factor (G-CSF) re-
ceptor, macrophage colony stimulating factor (M-CSF) receptor, granular macrophage colony stimulating factor
55 (GM-CSF) receptor, tumor necrosis factor (TNF) receptor, interleukin-1 (IL-1 ) receptor, interleukin-2 (IL-2) receptor,
interleukin-3 (IL-3) receptor, interleukin-4 (IL-4) receptor, interleukin-5 (IL-5) receptor, interleukin-6 (IL-6) receptor,
interleukin-7 (IL-7) receptor, interleukin-9 (IL-9) receptor, interleukin-1 0 (IL-1 0) receptor, interleukin-1 1 (IL-11) re-
ceptor, interleukin-12 (IL-1 2) receptor, interleukin-1 3 (IL-1 3) receptor, interleukin-1 5 (IL-1 5) receptor, interferon-
112
EP 1 327 681 A1
alpha (IFN-alpha) receptor, interferon-beta (IFN-beta) receptor, interferon-gamma (IFN-gamma) receptor, growth
hormone (GH) receptor, insulin receptor, blood stem cell proliferation factor (SCF) receptor, vascular endothelial
growth factor (VEGF) receptor, epidermal cell growth factor (EGF) receptor, nerve growth factor (NGF) receptor,
fibroblast growth factor (FGF) receptor, platelet-derived growth factor (PDGF) receptor, transforming growth factor-
beta (TGF-beta) receptor, leukocyte migration inhibitory factor (LIF) receptor, ciliary neurotrophic factor (CNTF)
receptor, oncostatin M (OSM) receptor, Notch family receptor, E2F, E2F/DP1 or TAK1/TAB1 .
17. The modified antibody of any one of claims 1 to 16, wherein the agonist action is apoptosis induction, cell prolif-
eration induction, cell differentiation induction, cell division induction or cell cycle regulation action.
18. The modified antibody of any one of claims 1 to 17, wherein the modified antibody is mono-specific modified
antibody.
19. The modified antibody of any one of claims 1 to 17, wherein the modified antibody is multi-specific modified anti-
body.
20. The modified antibody of claim 19, wherein the modified antibody is bi-specific modified antibody.
21. The monoclonal antibody of claim 20, wherein the L chain V region and the H chain V region are from the same
monoclonal antibody.
22. The monoclonal antibody of any one of claims 1 to 21 which shows an equivalent or better agonist action (ED50)
compared with the parent monoclonal antibody.
23. The monoclonal antibody of claim 22 which shows at least 2-fold agonist action (ED50) compared with the parent
monoclonal antibody.
24. The monoclonal antibody of claim 23 which shows at least 1 0-fold agonist action (ED50) compared with the parent
monoclonal antibody.
25. The monoclonal antibody of any one of claims 1 to 21 which is derived from a parent antibody having substantially
no agonist action.
26. A compound comprising two or more H chain V regions and two or more L chain V regions of monoclonal antibody
and showing an equivalent or better agonist action (ED50) compared with a natural ligand that binds to a cell
surface molecule(s) or intracellular molecule(s).
27. The compound of claim 26 which shows at least 2-fold agonist action (ED50) compared with a natural ligand that
binds to a cell surface molecule(s) or intracellular molecule(s).
28. The compound of claim 27 which shows at least 1 0-fold agonist action (ED50) compared with a natural ligand that
binds to a cell surface molecule(s) or intracellular molecule(s).
29. The modified antibody or compound of any one of claims 1 to 28 which has substantially no intercellular adhesion
action.
30. The modified antibody or compound of any one of claims 1 to 28 which has intercellular adhesion action (ED50)
not more than 1/10 compared with the parent antibody.
31 . A DNA which encodes the modified antibody or compound of any one of claims 1 to 28.
32. An animal cell which produces the modified antibody or compound of any one of claims 1 to 28.
33. A microorganism which produces the modified antibody or compound of any one of claims 1 to 28.
34. Use of the modified antibody or compound of any one of claims 1 to 28 as an agonist.
35. A method of inducing an agonist action to cells which comprises administering the first ligand and the second
113
EP 1 327 681 A1
ligand that bind to a cell surface molecule(s) or intracellular molecule(s) and administering a substance that binds
to the first and the second ligands and crosslinks the first and the second ligands.
36. The method of claim 35 wherein the first and the second ligands are the same or different single chain Fv monomers.
5
37. The method of claim 35 or 36 wherein the substance that crosslinks the ligands is an antibody, an antibody fragment
or a bivalent modified antibody.
38. A method of causing agonist action to cells by crosslinking a cell surface molecule(s) or intracellular molecule(s)
10 using the modified antibody or compound of any one of claims 1 to 28.
39. The method of claim 38 wherein the agonist action is apoptosis induction, cell proliferation induction, cell differen-
tiation induction, cell division induction or cell cycle regulation action.
15 40. A medicine comprising as active ingredient the modified antibody or compound of any one of claims 1 to 29.
41 . Use of the modified antibody or compound of any one of claims 1 to 29 as medicine.
42. A method of screening a modified antibody comprising two or more H chain V regions and two or more L chain V
20 regions of antibody and showing an agonist action by crosslinking a cell surface molecule(s) or intracellular mol-
ecule^) which comprises the steps
(1) to produce a modified antibody comprising two or more H chain V regions and two or more L chain V
regions of antibody and binding specifically to a cell surface molecule(s) or intracellular molecule(s),
25 (2) to subject cells expressing said cell surface molecule(s) or intracellular molecule(s) to react with the mod-
ified antibody and
(3) to measure the agonist action in the cells caused by crosslinking said cell surface molecule(s) or intracellular
molecule(s).
30 43. A method of measuring an agonist action of a modified antibody comprising two or more H chain V regions and
two or more L chain V regions of antibody and showing an agonist action by crosslinking a cell surface molecule
(s) or intracellular molecule(s) which comprises the steps
(1) to produce a modified antibody comprising two or more H chain V regions and two or more L chain V
35 regions of antibody and binding specifically to a cell surface molecule(s) or intracellular molecule(s),
(2) to subject cells expressing said cell surface molecule(s) or intracellular molecule(s) to react with the mod-
ified antibody and
(3) to measure the agonist action in the cells caused by crosslinking said cell surface molecule(s) or intracellular
molecule(s).
40
44. A method of producing a modified antibody comprising two or more H chain V regions and two or more L chain V
regions of monoclonal antibody and showing an agonist action by crosslinking a cell surface molecule(s) or intra-
cellular molecule(s) which comprises the steps
45 (1 ) to culture animal cells of claim 32 or microorganisms of claim 33 to produce the modified antibody and
(2) to purify said monoclonal antibody.
50
55
114
EP 1 327 681 A1
Fig. 1
Fig. 2
115
EP 1 327 681 A1
Fig. 3
o 980727.007
R.1 RTC
116
EP 1 327 681 A1
117
EP 1 327 681 A1
118
EP 1 327 681 A1
Fig . 6
119
EP 1 327 681 A1
Fig. 7
kDa
marker
120
EP 1 327 681 A1
Fig. 8
Fig. 9
990128.053
990128.054
121
EP 1 327 681 A1
Fig. 10
o 990128.057
FL1 RTC
Fig. 11
122
EP 1 327 681 A1
Fig. 12
Competitive ELISA
O.0O1
Dilution ratio of CM
pCH01/COS7 CM
MABL2-3cFv/COS7 CM
Fig. 13
990209.001
10
AnnexJn V-HTC
123
EP 1 327 681 A1
Fig. 14
Fig. 15
124
EP 1 327 681 A1
Fig. 16
990209.007
Annexin V-RTC
Fig. 17
990128.006
Annexln V-FITC
125
EP 1 327 681 A1
Fig. 18
990128.008
Annaxin V-FITC
126
EP 1 327 681 A1
Fig. 19
elution time (min)
127
EP 1 327 681 A1
128
EP 1 327 681 A1
Fig. 21
SDS-PAGE analysis of MABL2-scFv
<CHO>
<E. coli>
0>
E
kDa
97.
45-*-
31->-
£
o
c
o
£
©
E
<0
E
o
c
o
E
21 • 5_ *" ^^^8
L
+2ME -2ME
kDa
116-*-^
97,4 3III
E
o
c
o
E
03
£
0)
£
o
c
o
-45-
31-
215-
145-
1111
I
mm
JL
+2ME -2ME
129
EP 1 327 681 A1
Fig. 22
TSK gel G3000SW
20 mM Acetate buffer, 0.15 M NaCI, pH 6.0
05
monomer §
dimer
CO OJ
QQ
±d XL
CM
CD CO
I
130
EP 1 327 681 A1
131
Fig. 25
EP 1 327 681 A1
990319.004
:
-
•
-. . • . -•• .
" -
3.80%
e r.*
.** •*""
. ' 2.76%
-a
3 t-
10"
10'
10 2
Annexin V-FITC
10
JO
Fig. 26
1
2
a.
&
a.
°24
10 u
990819.024
10 1
' 1 " ' "I" r—r
NT
Annexin V-FITC
20.09%
32.60%
10*
133
EP 1 327 681 A1
Fig. 27
990810.024
"8
CL
a
o . ^_
10"
)•;••
■ ■
AnnexinV-RTC
1 5.17%
39.97%
10
lor
10 1
Fig. 28
134
EP 1 327 681 A1
Fig. 29
135
EP 1 327 681 A1
Fig. 30
Annean V-FTTC
Fig. 31
o
2
P
a.
990925.036
•J /.^'-ii'/ • " ',. 53.30%
:is . s :'. _r --. T :
35.25%
10'
10 2
Annodn V-F1TC
10
10
136
EP 1 327 681 A1
Fig. 32
Effect ofMABL-2 (scFv) on serum hlgG
in KPMM2 i.v. SCID mice
PBS(-)
scFv/CHO monomer
scFv/CHO dimer
2500 5000 7500
Serum hlgG (jig/mL)
10000
p<0.01
137
EP 1 327 681 A1
Fig. 33
Effect of MABL-2 (scFv) on survival
of KPMM2 i.v. SCID mice
100
75
-*-f
ca
(d
>
*>
^ 25
10
20 30 40
Days after cell inoculation
scFv/CHO Dimer **
scFv/CHO Monomer
PBS(-)
50
;■ P<0.01 by t-test
138
EP 1 327 681 A1
Fig. 34
139
EP 1 327 681 A1
Fig. 35
140
EP 1 327 681 A1
Fig. 36
Base Sequence and Amino Acid Sequence of Linker for HL Type
Heavy chain
Light chain
• ■ • gtc teg agt
V S S
linker
gac gtc gtg •••
D V V
Plasmid
Number of
linker amino acid
linker .
FLAG
CF2HL-0fcCOSl
CF2HL-34jCOS1
CF2HL-4/pCOSl
CF2HL-54>COSl
CF2HL-6/pCOSl
CF2HL-7/pCOSl
0
3
7 -
gtc teg agt gac gtc gtg
V S S D V V
gtc teg agt ggt ggt tec gac gtc gtg
V S S G G S DVV
gtc teg agt ggt ggt ggt tec gac gtc gtg
VSSGGGS DVV
gtc teg agt ggt ggt ggt ggt tec gac gtc gtg
VSSGGGGS DVV
gtc teg agt gt ggt ggt ggt ggt tec gac gtc gtg
VSSGGGGGS DVV
gtc teg agt ggt ggt ggt ggt- ggt ggt tec gac gtc gtg
VSS GGGGGGSDVV
141
EP 1 327 681 A1
Fig. 37
142
EP 1 327 681 A1
Fig. 38
Base Sequence and Amino Acid Sequence of Linker for LH Type
Li^ht chain
Heavy chain fHf
••• gag ata aaa linker cag gtc caa ■•■ FLAG
E I K Q V Q
Pla3mid
Number of
linker amino acid
liakec
CF2LH-(tfpCOSl
0
gag ata aaa
cag gtc caa
E IK
Q V Q"
CF2LH-3/pCOSl
3
gag ata aaa tec gga ggc
cag gtc caa
E I K S G G
Q V Q
CF2LH-4/pCOSl
4
gag ata aaa tec gga ggt gge
cag gtc caa
E I K S G G G
Q V Q
CF2LH-5/pCOSl
5
gag ata aaa tec gga ggt ggt ggc
cag gtc caa
E IKS GGGG
Q V Q
CF2LH-6/pC0Sl
6
gag ata aaa tec gga ggt ggt ggt ggc
cag gtc caa
E IKSGGGGG
Q V Q
CF2LH-74>C0S1
7
gag ata aaa tec gga ggt ggt ggt ggt ggc
cag gtc caa
E IKSGGGGGG
Q V Q
143
EP 1 327 681 A1
Fig. 39
CO
CO
CO
CO
CO
CO
CO
CO
co
CO
CO
CO
O
o
O
o
o
o
o
O
o
o
o
o
O
o
a
o
o
o
o
o
o
o
O
O
.a.
.a.
a
a
Q.
Q.
a.
Q.
_a
.a
Q.
o
CO
to
<S
o
?5
L?3
co
J]
Jj
Jj
i
—j
■
I
X
i
X
1
X
t
X
■
I
i
X
X
X
X
X
X
X
-J
-J
_1
_J
_i
CM
CM
pj
CM
CM
CM
li.
CM
CM
cm
CM
u.
U.
u.
U.
u_
u.
U.
u.
u.
o
o
O
o
o
o
O
o
o
O
o
>
LL
CM
o
X
o
Q.
^^ffi^^^^R — sc(Fv)2
- '• .'V..' ■• : • ■ "-v.
144
EP 1 327 681 A1
Fig. 40a
145
EP 1 327 681 A1
Fig. 40b
RTC
CF2LH-6/pCOS1 COS7 CM
CF2LH-7/pCOS1 COS7
£
CF2LH-3/pCOS1 COS7 CM ™1
146
EP 1 327 681 A1
fr £H e CH 01 .01 Q 0l G 0l OV ,01 Q 0l
ap!PO| Xunpidcud aptpoitunipjdaid
147
EP 1 327 681 A1
Fig. 41b
CF2ML-4/pCOSt C0S7 CM
O
•a ~~
E°o -
a
a. o
°o
V;
4 ' - • * 24.74%
r -
r^r?
*^«. % , , 38.53%
"'• *
H8$&: '■•"<> •
10 u
io' to* *cr
Arwoxin V-FITC
NT
o
CF2LH-4/pCOS1 COS7 CM
CI
a o
Q- O.
25.67%
38.89%
i i 'mm I ii i i w i i i i "" T.
|2 <«3 iq*
"T7
10 1 10" 10'
Annexin V-FTTC
3 i~
CF2HL-6/pCOS1 C0S7 CM
:-: ' . 24.95%
^
f'C- 36.53%
•* JT* * * /
fponky.* .« * •»* . *
to" to' t<r to 15
Annexln V-FTTC
10
CF2LH-6/pCOS1 C0S7 CM
9 o
ft
a.
o
©
'V /V./' "V. 27.74%
- * ' 34.46%
10"
10* to' 10
Annexln V-F1TC
10"
CF2HL-7/pCOS1 C0S7 CM
CD O -
■a *-
1
a. o J
o
o
•". • i-.'V"
25.64%
30.31%
■ - » TTtwiri ■ i»»
10° 10* 10^
Annexln V-FITC
n n w |~ i i
10 3 10'
CF2lH-7/pCOS1 C0S7 CM
CO
» o
I
s
CL
e-
a. o
•
v" ' ' V ••
.-25.97%
,1,., , ,1 . ■ ■
K.-:- •* 35,05%
A|. .. . .
10
10' io' icr
Annexln V-FtTC
10"
148
EP 1 327 681 A1
Fig. 42
(A) pCOS1/L1210 cells (B) WAP/L1210 cells
149
EP 1 327 681 A1
Fig. 43
(A) h/AP/L1210 ceils (B) CCRF-CEM cefls
Binding Sites (nM). Binding Sites (nM)
— r-o — control m/gG
o — Intact MABL-2
— A — MABL2-scFv <HL-5>
0 — : MABL2-sc(Fv) 2
150
EP 1 327 681 A1
Fig. 44
0 2500 5000 7500 10000
Serum JVJ protein (ug/mL)
151
EP 1 327 681 A1
Fig. 45
100
75
50
25
-HL-5 10mg/kg *
HL-5 -Img/kg
HL-5 0.1mg/kg
Vehicle
100
20 40 60
Number of Days after Transplantation
152
EP 1 327 681 A1
Fig. 46
sc(Fv)2 10mg/kg*
sc(Fv)2 1mg/kg
sc(Fv)2 0.1mg/kg
Vehicle
■
■
i
0 20 40 60
Number of Days after Transplantation
153
EP 1 327 681 A1
154
EP 1 327 681 A1
Fig. 48
155
EP 1 327 681 A1
49
With Reducing Agent Without Reducing Agent
M:MW marker
1 :sc1 2B5 fractionA
2:sc1 2B5 fractionB
156
EP 1 327 681 A1
Fig. 50
290 142 67 32 12.4 kD
g (a) sc 12B5peakA I II I I
157
EP 1 327 681 A1
Fig. 51
E
a
O
CM
CD
I
E
c
o
in
0
o
c
CO
£3
o
V)
n
<
1.2
0.8
a 0.4
•hTpo
12B5 IgG
-J — i i i mil ■ ■ ■ ■ ■ '■'» i i ■■»""■ ■ i i t i mt 1 i i i i nit
0.0001 0.001
0.01 0.1
Concentration (nM)
10
100
Fig. 52
E
c
o
CM
CO
I
E
c
o
LO
<
(TJ
<D
O
c
to
a
o
w
n
<
1.5 r
1.0
0.5
0.0
-hTpo
-sc12B5 monomer
-sc12B5 dimer
i i i nun i
' ••"•> • ' " I ' I ' I I ,1 I I T III ■ tint
0.00001 0.0001 0.001 0.01 0.1 1
Concentration (nM)
10
100 1000
158
EP 1 327 681 A1
(ratio 8 Z) eoxreqaosq^
159
EP 1 327 681 A1
160
EP 1 327 681 A1
Fig. 5 5
Reductive condition
M 1 2 3 4
116kD —
97kD —
66 kD —
45 kD —
31 kD —
21 kD
14 kD —
Non-reductive condition
M I 2 3 4
^1111
ISPllllfll
M: Molecular weight marker
1 : SC12E1 0 fraction A
2: sc12E1 0 fraction B
3: db12E10 fraction C
4: db12E10 fraction D
161
EP 1 327 681 A1
Fig. 56
SC3 peak A
SC3 peakB
o
00
IN
<0
V
C
rt
JO
u
o
o
co
CM
<D
O
C
fO
JQ
M
O
a)
3
(scl2E10)
mAU
5.0
4.0
3.0
2.0
1.0
0.0
-1.0
0:67
0.0
mAU
60.0
40.0
20.0
0.0
0.0
10.0 20.0 30.0
Retention time (minute)
40.0
mm
20kD
41.24
J V
10.0
20.0
30.0
40.0
Retention time (minute)
162
EP 1 327 681 A1
Fig. 57
db3 Peak C
db3 peak D
o
00
o
o
C
ifl
a
o
00
%
o
00
CD
U
a
m
-Q
u
o
m
(dbl2E10 )
69 kD
mAU
SO
60
40
20
0
0.0 10.0 20.0 30.0
Retention time (minute)
40.0
mAU
30.0
20.0
10.0
0.0
41kD
0.0
10.0
20.0
30.0
40.0
mm
Retention time (minute)
163
EP 1 327 681 A1
Fig. 5 8
1 r
0.00001 0.0001 0.001 0.01 0.1 1 10
Concentration (nM)
164
EP 1 327 681 A1
INTERNATIONAL SEARCH REPORT
International application No.
PCT/JP01/09260
A. CLASSIFICATION OF SUBJECT MATTER
Int.Cl' C12N15/09, 15/52, C07K16/28, A61K39/395
According to International Patent Classification (IPC) or to both national classification and IPC
B. FIELDS SEARCHED
Minimum documentation searched (classification system followed by classification symbols)
Int.Cl 7 C12N15/09, 15/62, C07K16/28 , A61K39/395
Documentation searched other than minimum documentation to the extent that such documents are included in the fields searched
Electronic data base consulted during the international search (name of data base and, where practicable, search terms used)
JICST FILE (JOIS) , MBDLINE (STN) , WPI (DIALOG) , BIOSIS (DIALOG)
C. DOCUMENTS CONSIDERED TO BE RELEVANT
Category*
Citation of document, with indication, where appropriate, of the relevant passages
Relevant to claim No.
Y
Bijia DENG et al . , "An Agonist Murine Monoclonal
Antibody to the Human c-Mpl Receptor Stimulayes
Megakaryocytpoiesis" , Blood, 15 September, 1998, Vol.92,
No. 6, pages 1981 to 1988
US 5885574 A (Amgen Inc.),
23 March, 1999 (23.03.99),
& JP 2000-95800 A & EP 7739S2
& WO 96/03438 A
Bl
KIPRIYANOV et al . , "Bispecific CD3xCD19 Diabody for
T Cell -Mediated Lysis of Malignant Human B Cells",
Int. J. Cancer, (1998), Vol.77, No. 5, pages 763 to 772
WO O0/53S34 A (Chugai Pharmaceutical Co., Ltd.),
14 September, 2000 (14.09.00),
& EP 1167388 A
Ming-Hong XIB et al . , "Direct demonstration of MuSK
involvement in acetycholine receptor clustering through
identification of agonist ScFv" , Nature Biotechnology,
August, 1997, Vol.15, No . 8 , pages 768 to 771
1-44
1-44
1-44
1-44
1-44
Further documents arc listed in the continuation of Box C. \^} See patent family ann
* Special categories of cited documents; "T*
''A" document defining the general state of the art which is not
considered to be of particular relevance
"E" earlier document but published on or after the international filing "X"
date
"L" document which may throw doubts on priority clalm(s) or which is
cited to establish the publication date of another citation or other " Y"
special reason (ns specified)
"0" document referring to an oral disclosure, use, exhibition or other
means
l P" document published prior to the international fling date bat later "&"
than the priority date claimed
later document published after the international Tiling date or
priority date and not in conflict with the application but cited to
understand the principle or theory underlying the invention
document oTparticular relevance; the claimed invention cannot be
considered novel or canno! be considered to involve an iiivennve
step when the document is laken alone
document of particular relevance; the claimed invention cannot be
considered to involve an inventive step when the document is
combined with one or more other such documents, such
combination being obvious to a person skilled in the art
document member of the same patent family
Date
of the actual completion of the international search
29 January, 2002 (29.01.02)
Date of mailing of the international search report
05 February, 2002 (05.02.02)
Name and mailing address of the ISA/
Japanese Patent Office
facsimile No,
Authorized officer
Telephone No.
Form PCT/ISA/2I0 (second sheet) (July 1992)
165
EP 1 327 681 A1
INTERNATIONAL SEARCH REPORT
International application No.
PCT/JP01/09260
C (Continuation). DOCUMENTS CONSIDERED TO BE RELEVANT
Category*
Citation of document, with indication, where appropriate, of the relevant passages
Relevant to claim No.
A
HP 1035132 A (Chugai Pharmaceutical Co., Ltd.),
1-44
13 September, 2000 (13.09.00),
& WO 99/12973 A
Form PCT/ISA/210 (continuation of second sheet) (July 1992)
166
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